Age-related histopathological and biochemical testicular damages were ameliorated by vitamin C administration / Los daños testiculares histopatológicos y bioquímicos relacionados con la edad mejoraron con la administración de vitamina C

Introduction and objectives: Aging is an irreversible process associated with decreased biological functions that can lead to the reduction of reproductive organs capacities in males and females. Paternal age is a significant predictor of offspring health and development. So, the aim of this study was to evaluate the effects of vitamin C on histopathological and biochemical testicular changes following aging process with a focus on stereological methods. Material and methods: For this study, 48 adult male NMRI mice were divided into two control and experimental groups. Mice in experimental group were supplemented with vitamin C (150mg/kg) including 24-h interval by oral gavage for 33 weeks. Same regime was performed for animals in control group except that vitamin C was replaced by water. Then, right testes were extracted for stereological and left testes were used for molecular analyses on weeks 8, 12, and 33. (AU)

Introducción y objetivos: El envejecimiento es un proceso irreversible asociado a una disminución de las funciones biológicas que puede conducir a la reducción de la capacidad de los órganos reproductivos en hombres y mujeres. La edad paterna es un predictor significativo de la salud y el desarrollo de la descendencia. Por lo tanto, el objetivo de este estudio fue evaluar los efectos de la vitamina C sobre los cambios testiculares histopatológicos y bioquímicos posteriores al proceso de envejecimiento con un enfoque en los métodos estereológicos.Material y métodos: Para este estudio, 48 ratones NMRI machos adultos se dividieron en dos grupos de control y experimentales. Los ratones del grupo experimental se suplementaron con vitamina C (150mg/kg), incluido un intervalo de 24 horas mediante sonda oral durante 33 semanas. Se realizó el mismo régimen para los animales del grupo de control, excepto que se reemplazó la vitamina C por agua. Luego, se extrajeron los testículos derechos para estereología y los testículos izquierdos se utilizaron para análisis moleculares en las semanas 8, 12 y 33. (AU)

Photobiomodulation therapy reverses spermatogenesis arrest in hyperthermia-induced azoospermia mouse model.

Testicular heat stress leads to impairment of spermatogenesis in mammals. Involved mechanism in this vulnerability to heat-induced injury remains unclear, and research is being conducted to find an approach to reverse spermatogenesis arrest caused by hyperthermia. Recently, different studies have utilized photobiomodulation therapy (PBMT) therapy for the improvement of sperm criteria and fertility. This study aimed at evaluating the effect of PBMT on the improvement of spermatogenesis in mouse models of hyperthermia-induced azoospermia. A total of 32 male NMRI mice were equally divided into four groups consisting of control, hyperthermia, hyperthermia + Laser 0.03 J/cm2, and hyperthermia + Laser 0.2 J/cm2. To induce scrotal hyperthermia, mice were anesthetized and placed in a hot water bath at 43 °C for 20 min for 5 weeks. Then, PBMT was operated for 21 days using 0.03 J/cm2 and 0.2 J/cm2 laser energy densities in the Laser 0.03 and Laser 0.2 groups, respectively. Results revealed that PBMT with lower intensity (0.03 J/cm2) increased succinate dehydrogenase (SDH) activity and glutathione (GSH)/oxidized glutathione (GSSG) ratio in hyperthermia-induced azoospermia mice. At the same time, low-level PBMT reduced reactive oxygen species (ROS), mitochondrial membrane potential, and lipid peroxidation levels in the azoospermia model. These alterations accompanied the restoration of spermatogenesis manifested by the elevated number of testicular cells, increased volume and length of seminiferous tubules, and production of mature spermatozoa. After conducting experiments and analyzing the results, it has been revealed that the use of PBMT at a dosage of 0.03 J/cm2 has shown remarkable healing effects in the heat-induced azoospermia mouse model.

Insulin Ameliorates Folliculogenesis in An Experimental Model of PCOS Mice.

BACKGROUND: Insulin is an essential factor that controls female reproductive system. Insulin signaling via Foxo1 and Akt1 can improve steroidogenesis, cell proliferation, and protein synthesis. We aimed to determine the effect of insulin on possible changes in gene expression, hormonal status, and histological aspects of the ovary following the induction of the animal model of polycystic ovary syndrome (PCOS). MATERIALS AND METHODS: In this experimental study, 24 adult female NMRI mice weighing 25-30 g were randomly placed in three groups: control, PCOS (60 mg/kg dehydroepiandrosterone (DHEA) for 20 days, and PCOS+insulin (60 mg/kg DHEA for 20 days+100 µL insulin diluted in water twice a week for 30 consecutive days). Blood specimens were obtained from the heart and the serum levels of testosterone, progesterone, and estradiol were measured. Right, and left ovaries were removed for real-time polymerase chain reaction (PCR) and stereological study. RESULTS: DHEA injection significantly amplified the concentration of testosterone, progesterone, and estradiol. While insulin treatment amended the level of reproductive hormones. DHEA injection significantly reduced the expression levels of Irs1-4, Pdk1, Pi3k, and Akt1-3 and raised the expression level of Caspase-3. However, insulin administration amplified expression levels of Irs1-4, Pdk1, Pi3k, and Akt1-3, and reduced Caspase-3. The total volume of ovarian tissue in mice receiving DHEA significantly declined compared to the control group. Besides, a substantial decrease was detected in the number of ovarian antral, Graafian, and primordial follicles and also in the total number of corpus luteum following DHEA administration. Comparison of structural alterations in ovarian tissue between the PCOS+insulin and the PCOS groups displayed that insulin administration improved the total number of Graafian, primordial, and antral follicles and also corpus luteum. CONCLUSION: In general, short-term insulin treatment showed improvement in hormonal balance, folliculogenesis, and insulin resistance in the ovaries of the PCOS mice model.

Age-related histopathological and biochemical testicular damages were ameliorated by vitamin C administration.

INTRODUCTION AND OBJECTIVES: Aging is an irreversible process associated with decreased biological functions that can lead to the reduction of reproductive organs capacities in males and females. Paternal age is a significant predictor of offspring health and development. So, the aim of this study was to evaluate the effects of vitamin C on histopathological and biochemical testicular changes following aging process with a focus on stereological methods. MATERIAL AND METHODS: For this study, 48 adult male NMRI mice were divided into two control and experimental groups. Mice in experimental group were supplemented with vitamin C (150mg/kg) including 24-h interval by oral gavage for 33 weeks. Same regime was performed for animals in control group except that vitamin C was replaced by water. Then, right testes were extracted for stereological and left testes were used for molecular analyses on weeks 8, 12, and 33. RESULTS: Our findings showed low semen quality, decreased level of serum Luteinizing hormone (LH), Follicle-stimulating hormone (FSH), and testosterone along with increased reactive oxygen species (ROS) production and higher apoptotic gene expression following aging. Stereological studies showed that the volume of testes, the length of seminiferous tubules, and the number of spermatogenic and none-spermatogenic cells decreased significantly during aging. Also, vitamin C consumption for 33 weeks significantly improved biochemical and histological indices. The impact of aging on male reproduction seems to be inevitable worldwide. Therefore, the use of protective and preventive remedies conserving male fecundity is very important and based on our results, vitamin C is a beneficial candidate for improving age-related testicular changes due to aging process.

An elderberry-supplemented diet improves spermatogenesis in mice with busulfan-induced azoospermia.

CONTEXT: Approximately 40-50% of all infertility cases are due to male infertility, and one of the most important causes of infertility is azoospermia. AIMS: This study aimed to evaluate the potential effect of elderberry on the spermatogenesis process in the azoospermia mice model. METHOD: Thirty adult male mice were randomised into three groups: control; busulfan (45mg/kg); and busulfan+elderberry (2%), 6mL orally per animal. Sperm samples were collected from the tail of the epididymis, and testis specimens were also collected and then subjected to sperm parameters analysis, histopathological evaluation, reactive oxygen species (ROS), and glutathione (GSH) measurement to determine the mRNA expression and hormonal assay. CONCLUSIONS: It can be concluded that the elderberry diet may be considered a complementary treatment to improve the spermatogenesis process in busulfan-induced azoospermic mice. IMPLICATIONS: Considering some limitations, the elderberry diet can be an alternate option for improving testicular damage following chemotherapy.

Therapeutic Effects of Edaravone on Azoospermia: Free Radical Scavenging and Autophagy Modulation in Testicular Tissue of Mice.

Background: Chemotherapeutic agents such as cyclophosphamide and busulfan have been shown to have a negative impact on the spermatogenesis process. Based on this fact, the objective of this study was to investigate the effects of edaravone on spermatogenesis in busulfan-induced mice. Methods: Forty adult male mice were equally divided into the four groups: 1) control, 2) edaravone, 3) busulfan, and 4) busulfan + edaravone. Then, the sperm parameters, histopathological examinations, and serum levels of testosterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) were also assessed. Caspase-3, Beclin-1, and ATG-7 mRNA levels were also determined using real-time PCR. Results: Our results revealed that treatment of mice with edaravone in busulfan-induced azoospermia significantly improves sperm parameters, including total count, morphology, and viability (p<0.05). Furthermore, edaravone administration led to a significant increase in serum testosterone (p<0.0001) and FSH (p<0.001) levels, as well as testis weight (p<0.05) and volume (p<0.01). Edaravone also prevented a decrease in the number of testicular cells including spermatogonia (p<0.0001), primary spermatocytes (p<0.001), round spermatids (p<0.0001), Sertoli (p<0.01), and Leydig cells (p<0.0001) in busulfan-treated mice. Additionally, in busulfan-induced azoospermia, edaravone significantly reduced the percentage of sperm with immature chromatin (p<0.0001). Following treatment with edaravone, a decrease in reactive oxygen species (ROS) and an increase in glutathione (GSH) production were noted compared to busulfan-treated mice. Furthermore, caspase-3 (p<0.05), Beclin-1, and ATG-7 (p<0.001) genes expression decreased significantly in treatment groups compared to busulfan-induced azoospermia. Conclusion: According to our findings, edaravone can improve spermatogenesis in busulfan-induced azoospermia through free radical scavenging and autophagy modulation in testicular tissue.

Evaluation of the Effects of Human Bone Marrow Mesenchymal Stem Cells Conditioned Medium on Growth and Maturation of Mouse Ovarian Follicle after Vitrification.

The aim of this research study is to evaluate the effect of human bone marrow mesenchymal stem cells conditioned medium (hBMSCs-CM) on growth and maturation of mouse ovarian follicle, and embryonic development after vitrification. The hBMSCs were cultured, and the derived CM was collected, concentrated, and stored. 14-day-old mice ovaries were collected and randomly divided into vitrified and non-vitrified groups. Then their isolated preantral follicles were cultured for 12 days in α-MEM supplemented with different concentrations of CM (2.5, 5, and 7.5%). Finally, the growth and diameter of follicles, maturation of oocytes, hormone level, and embryo developmental rate were assessed. The results showed the antrum formation, oocyte maturation, and hormone secretion were significantly higher in the presence of 7.5% CM (p < 0.001). In the vitrified group, the developmental rate of follicles was lower than the non-vitrified group, and the subgroup containing 7.5% CM showed better results than the 5%, and 2.5% CM subgroups. However, no changes in fertilization and embryonic development rates were observed. Supplementing follicle culture media with 7.5% CM could enhance follicle growth and oocyte maturation of follicles after vitrification.

Impaired spermatogenesis caused by busulfan is partially ameliorated by treatment with conditioned medium of adipose tissue derived mesenchymal stem cells.

Busulfan (BSU) is a chemotherapeutic drug that can cause subfertility or sterility in males. We investigated the effects of adipose tissue-derived mesenchymal stem cells (AT-MSC) conditioned medium (CM) (AT-MSC-CM) on histopathological and molecular characteristics of mouse testes exposed to BSU using stereology. We used adult male mice divided randomly into five groups: control, Dulbecco's modified Eagle's medium (DMEM), dimethyl sulfoxide (DMSO), BSU, and BSU + CM. Thirty-five days following BSU injection, sperm and testis tissues were harvested for stereological and molecular studies. The BSU group exhibited significantly reduced testis volume, interstitium and tubules compared to the other groups, although the volume of the testis remained unchanged for BSU and CM groups. The number of testis cells was reduced in the BSU group compared to the other groups. The CM group exhibited a significantly increased number of testis cells compared to the BSU group. Sperm count and motility, and length density of seminiferous tubules were increased in CM group compared to the BSU group. AT-MSC-CM exhibited ameliorative effects on histopathologic changes of mouse testes exposed to BSU.

Non-apoptotic cell death such as pyroptosis, autophagy, necroptosis and ferroptosis acts as partners to induce testicular cell death after scrotal hyperthermia in mice.

Cell death is a biologically uncontrollable and regulated process associated with human diseases which usually occur in response to oxidative stress that activates signalling pathways in multiple forms and can therefore contribute to human diseases. Thus, the current study aims to evaluate the signalling pathway involved in cell death after testicular hyperthermia. For this purpose, 32 mice were equally divided into four groups; I: Control; II, III and IV, Scrotal hyperthermia in which the testes are exposed to water at 43°C for 20 min every other day, respectively, 15, 10 and 5 times. Then, animals were euthanized and testicular tissue samples were isolated to evaluate protein expression as well as germ cell gene marker expression by Western blot and real-time PCR tests. Our data showed that the protein expression of Caspase-1, Beclin1, Atg7, Mlkl and Acsl4 together with the expression of Caspase-1, Beclin1, Atg7, Mlkl and Acsl4 genes was significantly up-regulated in scrotal hyperthermia-induced mice. In conclusion, the present study showed that heat stress disrupts spermatogenesis by activating several non-apoptotic signalling pathways in testicular tissue.

COVID-19 disrupts the blood-testis barrier through the induction of inflammatory cytokines and disruption of junctional proteins.

OBJECTIVE: Junctional proteins are the most important component of the blood-testis barrier and maintaining the integrity of this barrier is essential for spermatogenesis and male fertility. The present study elucidated the effect of SARS-CoV-2 infection on the blood-testis barrier (BTB) in patients who died from severe acute respiratory syndrome coronavirus 2 (COVID-19) complications. METHODS: In this study, lung and testis tissue was collected from autopsies of COVID-19 positive (n = 10) and negative men (n = 10) and was taken for stereology, immunocytochemistry, and RNA extraction. RESULTS: Evaluation of the lung tissue showed that the SARS-CoV-2 infection caused extensive damage to the lung tissue and also increases inflammation in testicular tissue and destruction of the testicular blood barrier. Autopsied testicular specimens of COVID-19 showed that COVID-19 infection significantly changes the spatial arrangement of testicular cells and notably decreased the number of Sertoli cells. Moreover, the immunohistochemistry results showed a significant reduction in the protein expression of occluding, claudin-11, and connexin-43 in the COVID-19 group. In addition, we also observed a remarkable enhancement in protein expression of CD68 in the testes of the COVID-19 group in comparison with the control group. Furthermore, the result showed that the expression of TNF-α, IL1ß, and IL6 was significantly increased in COVID-19 cases as well as the expression of occludin, claudin-11, and connexin-43 was decreased in COVID-19 cases. CONCLUSIONS: Overall, the present study demonstrated that SARS-CoV-2 could induce the up-regulation of the pro-inflammatory cytokine and down-regulation of junctional proteins of the BTB, which can disrupt BTB and ultimately impair spermatogenesis.

COVID-19 disrupts spermatogenesis through the oxidative stress pathway following induction of apoptosis.

To evaluate the incidence of apoptosis within the testes of patients who died from severe acute respiratory syndrome coronavirus 2 (COVID-19) complications, testis tissue was collected from autopsies of COVID-19 positive (n = 6) and negative men (n = 6). They were then taken for histopathological experiments, and RNA extraction, to examine the expression of angiotensin-converting enzyme 2 (ACE2), transmembrane protease, serine 2 (TMPRSS2), BAX, BCL2 and Caspase3 genes. Reactive oxygen species (ROS) production and glutathione disulfide (GSH) activity were also thoroughly examined. Autopsied testicular specimens of COVID-19 showed that COVID-19 infection significantly decreased the seminiferous tubule length, interstitial tissue and seminiferous tubule volume, as well as the number of testicular cells. An analysis of the results showed that the Johnsen expressed a reduction in the COVID-19 group when compared to the control group. Our data showed that the expression of ACE2, BAX and Caspase3 were remarkably increased as well as a decrease in the expression of BCL2 in COVID-19 cases. Although, no significant difference was found for TMPRSS2. Furthermore, the results signified an increase in the formation of ROS and suppression of the GSH activity as oxidative stress biomarkers. The results of immunohistochemistry and TUNEL assay showed that the expression of ACE2 and the number of apoptotic cells significantly increased in the COVID-19 group. Overall, this study suggests that COVID-19 infection causes spermatogenesis disruption, probably through the oxidative stress pathway and subsequently induces apoptosis.

An effective method for establishing animal models of azoospermia and oligospermia.

The current study aims to develop a validated animal model to predict successful spermatogenesis retrieval in azoospermia and oligospermia men. Thirty-two mice were equally divided into 4 groups: control, scrotal hyperthermia (15 times), scrotal hyperthermia group (10 times), scrotal hyperthermia group (5 times). In the scrotal hyperthermia groups, their scrotum exposed to water at a temperature of 43°C for 20 min every other day. Then, the mice were euthanised and sperm samples were collected for sperm parameters analysis, and blood samples were obtained for hormonal assay. The testis samples were taken for histopathology experiments, immunofluorescence staining and Western blot in order to examine the protein expression together with RNA extraction in order to examine the gene expression of germ cell markers. The results of sperm analysis and histopathology of testicular tissue as well as the results of gene expression and Western blot showed that hyperthermia can significantly impair spermatogenesis. In conclusion, we have developed a novel model of azoospermia and oligospermia in mouse, which uses a high temperature to suppress spermatogenesis process through demolition of germ cells subsequent cell cycle arrest and apoptosis. The model will contribute to understanding azoospermia in human, oligospermia pathophysiology and the development of treatment.

Chronic scrotal hyperthermia induces azoospermia and severe damage to testicular tissue in mice.

Scrotal hyperthermia leads to altered spermatogenesis due to heat-related oxidative stress. One of the main causes of infertility in men is oxidative stress, which refers to an imbalance in the levels of reactive oxygen species (ROS) and antioxidants. Therefore, this study aimed to evaluate the effects of chronic scrotal hyperthermia on testicular tissue structure, sperm parameters, and oxidative stress in adult mice. Thirty adult NMRI male mice were divided into three groups: Control (n = 10), Sham (n = 10), and Hyperthermia (n = 10). At the end of the study animals were sacrificed for evaluations of biochemical, cellular and histological analysis. The Hyperthermia group revealed a significant reduction in sperm count and weight of testis when compared to the control and sham groups. Also, succinate dehydrogenase (SDH) activity, ROS, ATP production, glutathione disulfide (GSH), tiols metabolism and stereological parameters in the hyperthermia group showed a significant reduction compared to the control and sham groups. Our results also revealed that scrotal hyperthermia significantly increases ROS production, mitochondrial membrane permeability (MMP), malondialdehyde (MDA), oxidized glutathione (GSSG) and apoptotic cells in testicular tissue in the hyperthermia groups in comparison with the control and sham groups. Overall, our result indicated that chronic scrotal hyperthermia causes complete spermatogenic arrest, probably mainly throughout the induction of oxidative stress.

Photobiomodulation Therapy Improves Spermatogenesis in Busulfan-Induced Infertile Mouse.

About 50% of infertility is caused by men. This study aimed to investigate the efficiency of photobiomodulation on spermatogenesis in a busulfan-induced infertile mouse as a testicular degeneration treatment. Thirty-two adult NMRI male mice were divided into 4 groups: control, busulfan, PBMT 0.03 J/cm2, and laser 0.2 J/cm2. In the study, azoospermia was induced by busulfan as a testicular degeneration, and then, they were treated using photobiomodulation therapy at 0.03 J/cm2 and 0.2 J/cm2 energy densities. Sperm parameters, stereological analysis, serum testosterone levels, together with SDH activity, MDA production oxidized as a marker for lipid peroxidation, glutathione (GSSG) and glutathione (GSH), mitochondrial membrane permeability (MMP), reactive oxygen species (ROS) production, and ATP production as well as TUNEL assay were assessed. Photobiomodulation therapy with 0.03 J/cm2 energy densities group revealed a significant increase the testosterone hormone level and spermatogenic cells with the reduction of apoptotic cells and marked increase in GSH, ATP, and SDH levels and decrease the levels of MDA and ROS production in the busulfan-induced mice when compared with the control and sham groups. In conclusion, the photobiomodulation therapy (0.03 J/cm2 energy density) may provide benefits on the spermatogenesis following busulfan injection and might be an alternative treatment to the patients with oligospermia and azoospermia in a dose-dependent manner.

Curcumin-Loaded Iron Particle Improvement of Spermatogenesis in Azoospermic Mouse Induced by Long-Term Scrotal Hyperthermia.

Spermatogenesis process is sensitive to heat stress because the testicular temperature is 2 to 4 °C lower than the core body temperature. The current study aimed to investigate the effects of iron oxide nanoparticles containing curcumin on spermatogenesis in mice induced by long-term scrotal hyperthermia. In this experimental study, 18 mice were equally divided into the following three groups: control, scrotal hyperthermia, and scrotal hyperthermia + curcumin-loaded iron particles (NPs) (240 µL) (mice were treated for 20 days). Hyperthermia was induced by exposure to the temperature of 43 °C for 20 min every other day for 5 weeks. Afterward, the animals were euthanized; sperm samples were collected for sperm parameters analysis, and testis samples were taken for histopathology experiments, evaluation of serum testosterone level, and RNA extraction in order to examine the expression of c-kit, STRA8 and PCNA genes. Our study showed that curcumin-loaded iron particles could notably increase the volume of testis, length of seminiferous tubules, sperm parameters, and stereological parameters (i.e., spermatogonia, primary spermatocyte, round spermatid, and Leydig cells) thereby increasing serum testosterone level; in addition, TUNEL-positive cells showed a significant decrease in curcumin-loaded iron particle group. Thus, based on the obtained results, the expression of c-kit, STRA8, and PCNA genes was significantly increased in treatment groups by curcumin-loaded iron particles compared with scrotal hyperthermia-induced mice. In conclusion, curcumin-loaded iron particles can be considered an alternative treatment for improving the spermatogenesis process in scrotal hyperthermia-induced mice.

Ameliorating effects of curcumin-loaded superparamagnetic iron oxide nanoparticles (SPIONs) on the mouse testis exposed to the transient hyperthermia: A molecular and stereological study.

BACKGROUND: Testicular hyperthermia can have negative effects on male fertility. Despite reported therapeutic benefits of curcumin, several factors often limit its application such as low water solubility and instable structure. Curcumin-loaded superparamagnetic iron oxide nanoparticles (SPIONs) were designed to solve its limitation of use. In the present study, we evaluated the effect of curcumin-loaded SPIONs on transient testicular hyperthermia in mouse. MATERIALS AND METHOD: A total of 18 adult male NMRI mice were divided into three groups (n = 6): I. Controls (Cont), II. Scrotal hyperthermia (Hyp), III. Scrotal hyperthermia + curcumin-loaded iron particles (240 µL) (Hyp + Cur). After seventy days, the animals were sacrificed and used for further molecular and stereological evaluations. RESULTS: Sperm count, motility and viability significantly decreased in group hyp as compared to cont group. Furthermore, Sperm DNA fragmentation and cell apoptosis in testes increased remarkably in group hyp, compared with group cont. Stereological study showed a reduction in number of spermatogenic and Leydig cells, as well as reduced weight and volume of testes in hyp group. Degenerative appearance of testes exposed to hyperthermia was also observed. In addition, higher mRNA expression of inflammatory cytokines (IL1-α, IL6, and TNF-α) was detected in group hyp compared to cont group. However, curcumin-loaded SPIONs alleviated all of the pathologic changes in the Hyp + Cur group compared to the hyp group. CONCLUSION: Here, we used nanoparticle form of curcumin in testicular hyperthermia model and showed its ameliorating effects on testes damages caused by heat stress, which can be an appropriate method to overcome the problems that limit curcumin application in cases with increased intra testicular temperature.

The modified method of luxol fast blue for paraffin-embedded myelin sheath staining / El método modificado de luxol fast blue para la tinción de mielina fijada en parafina

SUMMARY: Histological techniques are the study of animal and human tissues through staining and examining them under a microscope. To demonstrate the axonal degeneration and demyelination in histological studies, the Luxol Fast Blue staining is gold standard techniques. In this study, a new histochemical method based on modified Luxol Fast Blue for the staining of the myelin sheath in sciatic nerve tissues described. The sciatic nerves of rats were removed and then the sciatic nerve was immersed in 10 % formaldehyde for one week and embedded in paraffin block. Next, thin sections (5 µm) were cut, using a microtome and stained with conventional and modified Luxol Fast Blue. Our results showed that a new method of modified Luxol Fast Blue staining can accurately identify the myelin in the sciatic nerve fibers. The current study showed that the Luxol Fast Blue combination with Light Green has a good effect on myelin coloration, and the results of this study are comparable to LFB combination with Sirius red.

RESUMEN: Las técnicas histológicas son el estudio de tejidos animales y humanos mediante tinción y examen bajo un microscopio. Para demostrar la degeneración axonal y la desmielinización en estudios histológicos, la tinción Luxol Fast Blue es una técnica estándar de oro. En este estudio, se describe un nuevo método histoquímico basado en Luxol Fast Blue modificado para la tinción de mielina en los tejidos del nervio ciático. Se seccionaron los nervios ciáticos de ratas y luego el nervio ciático se sumergió en formaldehído al 10 % durante una semana y se fijó en bloque de parafina. Posteriormente, se cortaron secciones delgadas (5 µm) usando un microtomo y se tiñeron con Luxol Fast Blue convencional y modificado. Nuestros resultados mostraron que un nuevo método de tinción Luxol Fast Blue modificado puede identificar con precisión la mielina en las fibras del nervio ciático. El estudio actual mostró que la combinación Luxol Fast Blue con Light Green es un buen efecto sobre la coloración de mielina, y los resultados de este estudio son comparables a la combinación LFB con Sirius red.

Evaluating the Effects of Different Concentrations of Human Follicular Fluid on Growth, Development, and PCNA Gene Expression of Mouse Ovarian Follicles.

Follicle culture in vitro provides a method for investigating stages of folliculogenesis that can lead to preserving fertility through cryopreservation techniques. This study aims to assess the effects of various concentrations of human follicular fluid (hFF) on growth, development, and expression of the proliferating cell nuclear antigen (PCNA) gene in mouse ovarian follicles in vitro. Preantral follicles were isolated from 14-day NMRI mouse ovaries. The follicles were cultured in basic media enriched with FBS, FSH, and insulin-transferrin-selenium, and supplemented with different concentrations of hFF (10, 20, and 30%) for 12 days. During the culture period, survival rate and follicular maturation, follicular diameter, levels of estrogen and progesterone secretion, and PCNA gene expression rate were evaluated. Survival rate, maturation, and antrum formation were significantly higher in the 10% hFF group than in the 20 and 30% hFF groups. On day 4, follicle diameter in the 10% hFF group was also higher than in the 20 and the 30% hFF group. In comparison with other groups, significantly higher estrogen and progesterone production levels were measured in the 10% hFF group. PCNA gene expression was also higher with 10 than 20 and 30% hFF concentrations. The present study suggests that addition of 10% hFF to mice ovarian preantral follicle culture media enhances follicle growth and oocyte maturation.

Spatial arrangement of testicular cells disrupted by transient scrotal hyperthermia and subsequent impairment of spermatogenesis.

The spermatogenesis is temperature-dependent and heat stress have destructive effects on spermatogenesis and reduces sperm quality. Sixteen adult mice were allocated to two groups: hyperthermia and control groups. Scrotal hyperthermia was induced by water bath with 43°C for 30 min. Then, the spermatozoon was isolated through the tail region of epididymis for sperm parameters analysis. The testicular tissues were taken for stereological studies, hormonal assay, TUNEL assay and molecular studies. We found a marked decrease in sperm parameters and serum testosterone level in mice induced by scrotal hyperthermia as well as stereological analysis indicated a significant reduction in testicular cells and changes in the spatial arrangement of testicular cells in the scrotal hyperthermia groups compared to the control groups. Moreover, the TUNEL assay results showed that apoptotic cells were enhanced significantly in the group of scrotal hyperthermia compared to the control groups. Furthermore, scrotal hyperthermia caused a reduction in the expression of retinoic acid 8 (STRA8), c-kit and proliferating cell nuclear antigen (PCNA) genes in the scrotal hyperthermia groups compared to the control. According to results, induction of transient scrotal hyperthermia leads to a fluctuation in the spatial arrangement of testicular cells, which finally influences the normal function of spermatogenesis.

Photobiomodulation restores spermatogenesis in the transient scrotal hyperthermia-induced mice.

OBJECTIVE: Heat stress shock affects the generation of free radicals and can have a harmful effect on spermatogenesis. Photobiomodulation (PBM) is very effective in andrology for treating male infertility. This research aimed at the evaluation of the impacts of PBM on spermatogenesis on the transient scrotal hyperthermia-induced oligospermia mouse model. MATERIALS AND METHODS: This experimental research divided 24 mice into the following four groups: (1) Control, (2) Scrotal hyperthermia, (3) Scrotal hyperthermia receiving laser 0.03 J/cm2 for 30 s for each testis, 35 days after induction of scrotal hyperthermia every other day for 35 days, and (4) Scrotal hyperthermia receiving laser 0.03 J/cm2 for 30 s for each testis, immediately after induction of scrotal hyperthermia every other day for 35 days. Scrotal hyperthermia was induced by water bath with 43 °C for 30 min. Then, the mice were euthanized, and their sperm samples were collected for sperm parameters analysis. Then, we took the testis samples for histopathological experimentations, serum testosterone level, reactive oxygen species (ROS), RNA extraction for the examination of IL1-α, IL6 and TNF-α genes expression as well as production and glutathione disulfide (GSH) activity. KEY FINDINGS: Our outputs indicated that PBM could largely improve the sperms parameters and stereological parameters, like spermatogonia, primary spermatocyte, round spermatid and Leydig cells together with an increasing level of the serum testosterone and GSH activity compared to the scrotal hyperthermia induced mice. In addition, it was found that the diameter of seminiferous tubules, ROS production, as well as the expression of IL1-α, IL6, and TNF-α genes significantly decreased in the treatment groups by PBM compared to the scrotal hyperthermia induced mice, but there was not a significant difference in terms of testis weight and Sertoli cells between the studied groups. SIGNIFICANCE: It could be concluded that PBM may be regarded as an alternative treatment for improving the spermatogenesis process in the scrotal hyperthermia induced mice.