Stem Leydig cells support macrophage immunological homeostasis through mitochondrial transfer in mice.

As testicular mesenchymal stromal cells, stem Leydig cells (SLCs) show great promise in the treatment of male hypogonadism. The therapeutic functions of mesenchymal stromal cells are largely determined by their reciprocal regulation by immune responses. However, the immunoregulatory properties of SLCs remain unclear. Here, we observe that SLCs transplantation restore male fertility and testosterone production in an ischemia‒reperfusion injury mouse model. SLCs prevent inflammatory cascades through mitochondrial transfer to macrophages. Reactive oxygen species (ROS) released from activated macrophages inducing mitochondrial transfer from SLCs to macrophages in a transient receptor potential cation channel subfamily member 7 (TRPM7)-mediated manner. Notably, knockdown of TRPM7 in transplanted SLCs compromised therapeutic outcomes in both testicular ischemia‒reperfusion and testicular aging mouse models. These findings reveal a new mechanism of SLCs transplantation that may contribute to preserve testis function in male patients with hypogonadism related to immune disorders.

Abundance of selected genes implicated in testicular functions in Camelus dromedarius with high and low epididymal semen quality.

Studying testicular genes' expression may give key insights into precise regulation of its functions that influence epididymal sperm quality. The current study aimed to investigate the abundance of candidate genes involved in the regulation of testicular functions specially those regulate sperm function (PLA2G4D, SPP1, and CLUAP1), testicular steroidogenic function (ESR1 and AR), materials transport (AQP12B and LCN15), and defense mechanisms (DEFB110, GPX5, SOCS3, and IL6). Therefore, blood samples and testes with epididymis were collected from mature middle-aged (5-10 years) dromedary camels (n = 45) directly prior and after their slaughtering, respectively, during breeding season. Sera were evaluated for testosterone level and testicular biometry was measured with caliper. The epididymal tail semen was evaluated manually. Samples were distinguished based on testosterone level, testicular biometry, as well as epididymal semen features into high and low fertile groups. Total RNA was isolated from testicular tissues and gene expression was done using Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR). Results revealed that testosterone levels were significantly (P < 0.005) higher in camels with good semen quality than those of low quality. There was a significant (P < 0.0001) increase in testicular weight, length, width, thickness, and volume in high fertile than low fertile camels. PLA2G4D, SPP1, CLUAP1, ESR1, AR, AQP12B, LCN15, DEFB110, GPX5, and SOCS3 genes were upregulated (P < 0.001), and IL6 gene was downregulated (P < 0.01) in the testes of high fertile camels compared to the low fertile one. Thus, it could be concluded that examined genes might be valuable monitors of testicular functional status and fertility in dromedary camels.

High cholesterol diet-induced testicular dysfunction in rats.

PURPOSE: Hypercholesterolemia due to a high-cholesterol diet is linked to numerous diseases and may lead to male infertility. However, the underlying mechanism remains unknown. The maintenance of male fertility requires intact testicular structures (including seminiferous tubules and mesenchyme) and functioning cells (Leydig cells, Sertoli cells and germ cells, etc.), production of appropriate concentrations of sex hormones, and cooperation among testicular cells. Thus, we considered whether male fertility declined as the structure and function of testicular cells were altered in rats on a high-cholesterol diet. METHODS: Male Sprague Dawley rats were fed either a standard or a high-cholesterol diet for 16 weeks. Serum sex hormones, lipid components, semen quality, and fertility rate were assayed in the rats. The 3ß-hydroxysteroid dehydrogenase (3ß-HSD), Wilms tumor 1 (WT-1), and deleted in azoospermia-like (DAZL) were regarded as specific markers of Leydig, Sertoli, and germ cells in rats. In addition, the ultrastructure of the testis and expression levels of particular marker molecules of testicular cells were further investigated. RESULTS: Compared to rats fed on a regular diet, the serum testosterone levels and sperm progressive motility decreased in rats fed high cholesterol. Moreover, we observed a deformed nucleus, dilated smooth endoplasmic reticulum, and swollen mitochondria of Leydig cells and a schizolytic nucleus of Sertoli cells in rats on a high-cholesterol diet. The 3ß-HSD, WT-1, and DAZL protein expression levels were significantly reduced in rats on a high-cholesterol diet. CONCLUSIONS: Our results showed that a high-cholesterol diet adversely affected testosterone production and sperm progressive motility, possibly due to Leydig, Sertoli, and germ cell abnormalities.

Effects of therapeutic ultrasound and moderate heat on stallion testes.

Transplantation of stem cells into dysfunctional testes is currently being investigated as a therapeutic option for men and stallions with advanced testicular degeneration. This series of "proof of concept" studies aimed to identify a safe and efficient method of inducing severe testicular degeneration to create an optimal equine recipient model for intratesticular stem cell transplantation (SCT). Two ex vivo and two in vivo experiments were conducted. At first, forty testes obtained from castrations were used to identify an effective therapeutic ultrasound (TUS) device and the protocol for increasing intratesticular temperature in stallions. Six min of treatment using the Vetrison Clinic Portable TUS machine raised the intratesticular temperature by 8°C-12.5 °C. This protocol was applied to treat three scrotal testes in three Miniature horse stallions, three times, every other day. Contralateral testes served as controls. There were signs of slight tubular degeneration in treated testes two and three weeks after TUS treatment. The number of seminiferous tubules (STs) with exfoliated germ cells (GCs) was increased in one testis only, three weeks after treatment. The degree of apoptosis of GCs was higher in each treated testis in comparison to the contralateral control testis. Next, the ability of various heating devices to increase intratesticular temperatures to at least 43 °C in stallion testes was tested, using twenty testes obtained from castrations. ThermaCare® Lower Back & Hip Pain Therapy Heatwrap (TC heat wrap) reliably increased intratesticular temperatures and kept them continuously between 43 °C and 48 °C for seven to 8 h. In the follow-up in vivo study, the left testes of three Miniature horse stallions were treated with TUS, after which both testes of each stallion were treated with moderate heat provided by the TC heat wrap (three times, every other day, for 5 h each time). There were signs of moderate tubular degeneration in the samples from all treated testes obtained three weeks after treatments (Heat only or Heat/TUS): areas with hypospermatogenesis, spermatogenic arrest, vacuolized Sertoli cells, numerous STs with exfoliated GCs, increased degree of GCs apoptosis, and changes in three histomorphometric numeric attributes of STs. We concluded that TUS or TC wraps increase intratesticular temperature of the isolated stallion testes. Further, treatment with TUS or moderate heat may induce mild to moderate degenerative changes in stallion testes. However, to achieve more robust result - severe testicular degeneration, our treatment protocol has to be modified.

Testicular torsion and subsequent testicular function in young men from the general population.

STUDY QUESTION: Is prior testicular torsion associated with testicular function (semen quality and reproductive hormones) in young men from the general population? SUMMARY ANSWER: In young men from the general population, no differences in semen parameters were observed in those who had experienced testicular torsion compared to controls and observations of higher FSH and lower inhibin B were subtle. WHAT IS KNOWN ALREADY: Testicular function may be impaired after testicular torsion, but knowledge is sparse and based on studies with small sample sizes and no control group or a less than ideal control group. STUDY DESIGN, SIZE, DURATION: A cross-sectional population-based study was carried out including 7876 young Danish men with unknown fertility potential, examined from 1996 to 2020. PARTICIPANTS/MATERIALS, SETTING, METHODS: All men (median age 19.0 years) had a physical examination, provided a blood and semen sample, and filled in a questionnaire including information about prior testicular torsion, birth, lifestyle and current and previous diseases. Markers of testicular function, including testis volume, semen parameters and reproductive hormones, were compared between men operated for testicular torsion and controls, using multiple linear regression analyses. MAIN RESULTS AND THE ROLE OF CHANCE: The average participation rate was 24% for the entire study period. In total, 57 men (0.72%) were previously operated for testicular torsion (median age at surgery 13.4 years) of which five had only one remaining testicle. Men with prior testicular torsion were more often born preterm (25% versus 9.5% among controls), and they had significantly higher FSH and lower inhibin B levels, and a lower inhibin B/FSH ratio than controls in crude and adjusted models. The association was mainly driven by the subgroup of men who had undergone unilateral orchiectomy. No differences in semen parameters were observed. LIMITATIONS, REASONS FOR CAUTION: A limitation is the retrospective self-reported information on testicular torsion. Also, results should be interpreted with caution owing to the high uncertainty of the observed differences. WIDER IMPLICATIONS OF THE FINDINGS: Overall, the results of our study are reassuring for men who have experienced testicular torsion, especially when treated with orchiopexy, for whom reproductive hormone alterations were subtle and without obvious clinical relevance. Our study found no differences in semen parameters, but follow-up studies are needed to assess any long-term consequences for fertility. STUDY FUNDING/COMPETING INTEREST(S): Financial support was received from the Danish Ministry of Health; the Danish Environmental Protection Agency; the Research fund of Rigshospitalet, Copenhagen University Hospital; the European Union (Contract numbers BMH4-CT96-0314, QLK4-CT-1999-01422, QLK4-CT-2002-00603, FP7/2007-2013, DEER Grant agreement no. 212844); A.P. Møller and wife Chastine Mckinney Møllers Foundation; Svend Andersens Foundation; the Research Fund of the Capital Region of Denmark; and ReproUnion (EU/Interreg). The authors have nothing to declare. TRIAL REGISTRATION NUMBER: N/A.

Photoperiod alters testicular methyltransferase complex mRNA expression in Siberian hamsters.

Exposure to long photoperiods stimulates, whereas exposure to short photoperiods transiently inhibit testicular function in Siberian hamsters via well-described neuroendocrine mechanisms. However, less is known about the intra-testicular regulation of these photoperiod-mediated changes. N6-methyladenosine (m6A) is one of the most common mRNA modifications in eukaryotes, with alterations in m6A mRNA methylation affecting testis function and fertility. We hypothesized that genes controlling m6A methylation such as methyltransferase-like-3 (Mettl3) and -14 (Mettl14) and Wilms' tumor-1 associated protein (Wtap), part of an mRNA methylating methyl-transferase complex, or the fat-mass-and-obesity-associated (Fto) and the α-ketoglutarate-dependent dioxygenase alkB homolog-5 (Alkbh5) genes responsible for m6A demethylation, may be differentially regulated by photoperiod in the testis. Male hamsters were exposed to long (LD, control) photoperiod for 14-weeks, short (SD) photoperiod for 2, 5, 8, 11 and 14-weeks to induce regression, or SD for 14-weeks followed by transfer to LD for 1, 2, 4 or 8-weeks to induce recrudescence (post-transfer, PT). SD exposure significantly reduced body, testis, and epididymal masses compared to all other groups. Spermatogenic index, seminiferous tubule diameters and testosterone concentrations significantly decreased in SD as compared to LD, returning to levels no different than LD in post-transfer groups. SD exposure significantly decreased Wtap, Fto, Alkbh5, but increased Mettl14 mRNA expression as compared to LD, with values in PT groups restored to LD levels. Mettl3 mRNA expression did not change. These results suggest that testicular recovery induced by stimulatory photoperiod is relatively rapid, and that the methyltransferase complex may play a role during photostimulated testicular recrudescence.

Intramuscular administration of l-arginine boosts testicular hemodynamics, plasma concentrations of testosterone and nitric oxide in heat-stressed rams.

The current study aimed to assess, for the first time, the effects of intramuscular injection of l-arginine (L-arg) on testicular hemodynamics, echogenicity, and plasma concentrations of testosterone, total antioxidant capacity, and nitric oxide (NO) in Ossimi rams. Twelve sexually matured heat-stressed rams were randomly assigned to one of two groups: the L-arg group (n = 6) received 5 mg/kg L-arg dissolved in 2 ml normal saline 0.9%, or the control group (n = 6) received merely 2 ml of normal saline 0.9%. Blood sampling, B-mode ultrasound assessment of the testicular parenchyma, and pulsed-wave Doppler ultrasound of the testicular artery for both right and left testis were performed immediately before 0 min and 1, 4, 24, 48, 72, and 168 h after L-arg or saline administration. In the L-arg group, resistive index (RI) and pulsatility index (PI) means were significantly lower compared to the control group at 4-168 h post-treatment. Plasma testosterone concentrations were higher (P < 0.05) at 4 h and onward in the L-arg treated compared to the control rams, the same for NO levels however its increase (P < 0.05) was observed as soon as 1 h post-treatment. In L-arg treated rams, NO concentrations were positively correlated to plasma testosterone concentrations (r = 0.7, p < .01), but negatively correlated to both RI and PI (r = -0.8 and -0.6, respectively, p < .01). In conclusion, l-arginine administration enhanced testicular blood flow and increased plasma testosterone and nitric oxide concentrations in heat-stressed rams.

Diurnal rhythms in testicular blood flow, testicular morphometry and reproductive hormones in Shiba goats.

CONTEXT: Testicular blood flow (TBF) is crucial for testicular function. The pattern of TBF in Shiba goats indicates seasonal variations. AIMS: This study aimed to investigate the effect of diurnal variations on TBF, testis volume (TV), testicular echogenicity, and reproductive hormones in goats over a 24-h period. METHODS: In three trials that went for three consecutive days each, 12 bucks were scanned using Triplex ultrasonography to assess the TV, pixel intensity of testicular echotexture (PIX), and Doppler indices of TBF (resistive index: RI and pulsatility index: PI) in four-time points a day (at 6.00, 12.00, 18.00, and 00.00h). Concomitantly, the changes in circulating FSH, LH, inhibin, testosterone (T), estradiol (E2), cortisol, and melatonin were assessed. KEY RESULTS: Results revealed diurnal alterations in the calculated RI of TBF and the PIX of testicular parenchyma (P <0.05). Lower RI values of the TBF were observed at 6.00h compared to other time points. There were significant diurnal alterations in the levels of FSH (P <0.05), LH (P <0.05), T (P <0.0001), E2 (P <0.0001), cortisol (P <0.0001), and melatonin (P <0.0001). FSH attained a higher concentration at 18.00h compared to 12.00h. Concentrations of LH were significantly higher at 06.00h compared to those at 18.00h. Concentrations of T were significantly higher at 6.00 compared to other time points. E2 showed higher concentrations at 6.00h and 00.00h compared to 12.00h and 18.00h. On the contrary, concentrations of cortisol were significantly higher at 12.00h and 18.00h compared to 06.00h and 00.00h. The highest concentrations of melatonin were observed at 00.00h compared to other time points, while the lowest concentrations were at 12.00h. CONCLUSIONS: Diurnal rhythm induces significant changes in TBF, testicular PIX, and circulating FSH, LH, T, E2, cortisol, and melatonin over the 24-hday. IMPLICATIONS: The outcomes of the study are reflected in the advisability of monitoring the TBF at a fixed time a day to avoid the circadian rhythm effect.

Estrogen-related receptor is critical for testicular mitochondrial homeostasis and sperm motility: a Drosophila-based study.

OBJECTIVE: To study the role of estrogen-related receptors (ERRs) in testicular function, with particular emphasis on mitochondrial homeostasis, testicular steroidogenesis, and sperm motility using Drosophila as a model. DESIGN: Experimental study. SETTING: Academic research laboratory. ANIMAL(S): Wild-type and transgenic strains of Drosophila melanogaster. INTERVENTION(S): Using a ribonucleic acid interference-based approach, ERR was knocked down specifically in the testes to generate Drosophila males with reduced ERR levels in their testes. Genetically matched sibling males without the knockdown formed the controls. MAIN OUTCOME MEASURE(S): Analysis of the testicular mitochondrial structure and function in relation to energy production, steroidogenesis, and sperm motility in Drosophila. RESULT(S): Depletion of ERR affects mitochondrial homeostasis (biogenesis, fission, fusion, mitophagy, and transport) and oxidative respiration in the testes. Consequently, ERR knockdown testes have significantly reduced mitochondrial size, mass, and depleted adenosine triphosphate levels resulting in testicular oxidative stress. Further, Halloween genes, associated with steroidogenesis in Drosophila, are misregulated in ERR knockdown testes, and knockdown of some of the steroidogenic genes in a testis-specific manner results in significantly reduced fertility. In addition, sperm from ERR knockdown testes have significantly reduced levels of glucose transporter, Na+K+ ATPase, Dynein heavy chain, and adenosine triphosphate-5α synthase essential for sperm function. Corroborating this, sperm from ERR knockdown males are significantly less motile compared with control. CONCLUSION(S): The ERR is crucial for meeting the cellular energy requirements of the testes and the generation of normal motile sperm and hormone synthesis/secretion in the testes. To our knowledge, this is the first report implicating ERR in these ultimate functions of the testes. These findings can potentially contribute to the etiologic understanding of asthenozoospermia or infertility at large in men.

Effects of an anti-gonadoliberin releasing hormone vaccine on testicular, epididymal and spermatogenic development in the horse.

The effects of the GnRH vaccine Improvac® on testicular and epididymal morphometrics, histology and spermatogenesis were measured in 19 young (15-20 months) colts randomly assigned to one control (saline, castration at 57 days, n = 6) or either of two GnRH vaccine-treatment groups, T-57 (castration at 57 days, n = 7) or T-100 (castration at 100 days, n = 6), respectively. All were immunized on Day 0 with a single booster on Day 28. Excised testes and epididymides were weighed and processed for histology to measure tubule, epithelial and muscle dimensions, the ratio of interstitial tissue to seminiferous tubules and determine the stage of spermatogenesis. Testis volume, unchanged within controls, decreased in T-57 and T-100 groups by 50% and 70%, respectively. Treated colts' testes were significantly lighter than controls (64% relative difference); however, epididymal mass showed no significant differences between groups. Proportionally less seminiferous tubule relative to interstitial tissue was observed in both treatment groups (5%) versus controls (22%) with a mean tubule size 28% smaller than controls. Controls exhibited a high proportion of seminiferous tubules with advanced stages of spermatogenesis, whereas treated colts showed a high proportion of tubules in the early stages of spermatogenesis. In conclusion, immunization against GnRH in prepubertal colts was effective at reducing the development of their intra-scrotal reproductive organs and preventing normal spermatogenesis. GnRH vaccination of young colts effectively and consistently reduced testis mass, tubule size and relative proportion of seminiferous tubule tissue while retarding spermatogenesis. The epididymis showed changes with a smaller tubule diameter, lower epithelial height and thicker muscle layer recorded in treated compared to control colts.

Testicular haemodynamics, plasma testosterone and oestradiol concentrations, and serum nitric oxide levels in the Egyptian buffalo bull after a single administration of human chorionic gonadotropin.

This current study aimed for the first time to evaluate the effect of a single intravenous administration of human chorionic gonadotropin hormone (hCG) on the testicular artery haemodynamics measurements (resistance [RI], and pulsatility indices [PI]), plasma steroids (estradiol-17ß and testosterone) and nitric oxide (NO) levels in buffalo bulls. Twelve Egyptian buffalo bulls weighted 450 ± 20 kg were randomly divided into the hCG group (n = 6) and injected a single dose with Ovogest (EPIFASI; 5,000 IU, iv), whereas the others (n = 6) were injected with normal saline and served as controls. Doppler evaluation and blood sampling were performed just before the administration (hour 0) and at 1, 2, 4, 6, 8, 10, 24 and 28 hr after administration. Bulls in the control group did not show any alterations in hormonal levels and blood flow parameters (p > .05). In the hCG group, RI was declined (p < .05) in 6 hr post-administration (0.31 ± 0.01 versus 0.45 ± 0.01), while PI was declined (p < .05) later in 10 hr (0.74 ± 0.01 versus 1.23 ± 0.02). Additionally, testicular blood flow was increased (p < .05) 8 hr (42.02 ± 1.02 ml/min/100 g versus 31.34 ± 0.88 ml/min/100 g) after administration. Testosterone and NO levels were (p < .05) increased at 4 and 6 hr post-administration (3.55 ± 0.03 ng/ml versus 2.84 ± 0.01 ng/ml, and 55.32 ± 4.25 µmol/L versus 32.21 ± 1.55 µmol/L), whereas oestradiol levels were elevated (p < .05) in 6 hr (31.25 ± 0.08 pg/ml) only post-administration then declined. In conclusion, the single intravenous administration of hCG triggered many alterations in the supratesticular artery vascularization and hormonal profile that could affect positively on steroidogenesis and testicular function in buffalo bull.

Immunocastration with gene vaccine (KISS1) induces a cell-mediated immune response in ram testis: A transcriptome evaluation.

Immunocastration vaccines achieve their effects through neutralization of the endogenous hormone by the humoral antibody produced against the immunized genes, but there is little information regarding cell-mediated immune response on the gonadal function of the immunized model is available. In this study, we used ram as a model animal to identify the cellular immune response in testicular tissues of rams immunized with intranasal KISS1 gene vaccine. The immune castration model was evaluated by sexual behaviours, spermatogenesis and serum hormone profiles after the KISS1 gene immunization. Transcriptome analysis of testicular tissues was carried out to identify the expressions of protein-coding genes involved in cellular immunity. The results showed that we successfully constructed the KISS1 immune castration ram model, in which testicular growth and development, testosterone and kisspeptin-54 levels, and sexual function were suppressed in immunized rams (p < .05). Using Hiseq™ 2000 high sequencing for ram testicular, we identified 21 differentially expressed genes (DEGs) related to cellular immunity, of which, 14 genes were upregulated and seven genes were downregulated in the testis of the immunized group (p < .05). The Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment showed that these differentially expressed genes were enriched in the antigen presentation process mediated by MHC class I and the cytotoxic pathway mediated by natural killer cells. It is concluded that KISS1 gene vaccine induced the cell-mediated immune response in testicular tissue to suppress reproductive activities in rams.

Vitamin E rescues valproic acid-induced testicular injury in rats: Role of autophagy.

AIMS: Valproic acid (VPA), a commonly used antiepileptic drug, can induce testicular oxidative stress and injury. Altered autophagic response usually follows testicular injury. The study aims to evaluate the role of autophagy in the protective effect of the antioxidant vitamin E (Vit E) against VPA-induced testicular injury. MATERIALS AND METHODS: VPA (100, 300, and 500 mg/kg/day) was administered for 8 days. The protective group received both Vit E (50 mg/kg) and VPA (500 mg/kg). The testicular weight, sperm analysis, and serum testosterone concentration, as well as testicular histopathology, steroidogenic gene expression, and oxidative stress markers were evaluated. The mRNA or protein expression of autophagy-related proteins [adenosine monophosphate-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR), microtubule-associated protein light chain 3 (LC3), Beclin1, and p62] were measured using RT-PCR or immunohistochemistry. KEY FINDINGS: VPA resulted in lower testes weight and sperm quality with aberrant morphology. VPA dose-dependently induced testicular oxidative stress, which was associated with decreased steroidogenic gene expression and serum testosterone levels, as well as deteriorated histopathology. These biochemical and histological changes were also associated with autophagy induction (higher LC3 and Beclin1, and lower p62) that was lost with the highest toxic dose (500 mg/kg). The attenuated autophagy with the highest dose was accompanied by AMPK downregulation and mTOR upregulation. Vit E protected against VPA-mediated oxidative stress and toxicity while also restoring autophagic response and AMPK/mTOR levels. SIGNIFICANCE: The study highlights vitamin E as a valuable protective asset against VPA-induced testicular injury, possibly through AMPK-mTOR-dependent autophagy induction.

Immature Testicular Tissue Engineered from Weaned Mice to Adults for Prepubertal Fertility Preservation-An In Vivo Translational Study.

Male pediatric survivors of cancers and bone marrow transplantation often require adjuvant chemoradiation therapy that may be gonadotoxic. The optimal methods to preserve fertility in these prepubertal males are still under investigation. This manuscript presents an in vivo experiment which involved transplantation of immature testicular tissues (ITT) from transgenic donor, to wild-type recipient mice. Donors and recipients were age-mismatched (from 20-week-old donors to 3-week-old recipients, and vice versa) and the transplantation sites involved the abdomen, skin of the head, back muscle, and scrotum. The application of poly-l-lactic acid (PLLA) scaffold was also evaluated in age-matched donors and recipients (both 3-weeks-old). To quantitively evaluate the process of spermatogenesis after ITT transplantation and scaffold application, bioluminescence imaging (BLI) was employed. Our result showed that ITT from 3-week-old mice had the best potential for spermatogenesis, and the optimal transplantation site was in the scrotum. Spermatogenesis was observed in recipient mice up to 51 days after transplantation, and up to the 85th day if scaffold was used. The peak of spermatogenesis occurred between the 42nd and 55th days in the scaffold group. This animal model may serve as a framework for further studies in prepubertal male fertility preservation.

Zinc oxide nanoparticles attenuate prepubertal exposure to cisplatin- induced testicular toxicity and spermatogenesis impairment in rats.

Cisplatin exposure represents a significant fertility problem for childhood cancer. In this study we examined the possible therapeutic role of Zinc oxide nanoparticles (ZnO-NPs) on Cisplatin (Cis) induced impairment in the spermatogenesis initiation during puberty. Seventy-two male rats aged 30 days were distributed into four equal groups: Control group; ZnO-NPs group (intraperitoneal i.p. injected with 5 mg/kg ZnO-NPs once a week for eight weeks); Cis group (i.p. injected with a single dose of 5 mg/kg); ZnO-NPs + Cis group (ZnO-NPs injection 2 hrs before Cis). Each group was subdivided into three groups and was sacrificed 7, 30 and, 60 days after cisplatin induction, which considered prepubertal at 37-day-old, productive at 60-day-old, and completely adult at 90-day-old. Biochemical, molecular, immunohistochemical, and ultrastructural examinations were studied on the testicular tissues and sperm samples. Group treated with Cis showed a decrease in the antioxidant activity and an increase in the reactive oxygen species (ROS), which in turn caused disruption in blood-testis barrier (BTB) proteins in the three different rat ages, and sperm DNA damage in the adult rats compared to control group (p < 0.05). Moreover, alterations in the structural and the ultrastructural morphology of the testis were observed compared with the control at 37, 60 and 90 days old rats. ZnO-NPs administration to Cis group manifested a significant decrease in the ROS that restored the BTB proteins, enhanced the architecture of the testis in the three different rat ages, and increased sperm DNA integrity in the adult rats. Zinc oxide nanoparticles could restore the male reproductive capacity in the adult rats after induction of Cis in the prepubertal period by promoting spermatogenesis.

Effects of whole-life exposure to low-dose cadmium with post-weaning high-fat diet on offspring testes in a male mouse model.

Although several studies have reported testicular impairments caused by cadmium (Cd) or obesity alone, the combined effect of Cd and obesity on the testes and its underlying mechanism remains unclear. We examined the combined effect of whole-life exposure to low-dose Cd started at preconception and post-weaning high-fat diet (HFD) on the testes of offspring mice. At weaning, male offspring parented with and without exposure to low-dose Cd were continued on the same drinking water regimen as their parents and fed with either a normal diet (ND) or HFD for 10 or 24 weeks. Whole-life exposure to Cd resulted in its accumulation in testes, and HFD induced obesity and lipid metabolism disorder. Exposure to Cd or HFD alone significantly decreased Johnsen scores, disrupted testicular structure, and increased germ cell apoptosis at both 10 and 24 weeks. However, co-exposure to Cd and HFD did not induce the toxic effects that were induced by either alone, as revealed by preserved testicular structure and spermatogenesis, lack of significant apoptosis, and increased cell proliferation. Mechanistically, the combined effects of low-dose Cd and HFD consumption were associated with the activation of the JAK/STAT pathway. These findings suggest that co-exposure to low-dose Cd and HFD did not cause Cd- or HFD-induced testicular injury, probably because of the activation of the JAK/STAT pathway to prevent germ cell apoptosis.

Impact of cilia-related genes on mitochondrial dynamics during Drosophila spermatogenesis.

Spermatogenesis is a dynamic process of cellular differentiation that generates the mature spermatozoa required for reproduction. Errors that arise during this process can lead to sterility due to low sperm counts and malformed or immotile sperm. While it is estimated that 1 out of 7 human couples encounter infertility, the underlying cause of male infertility can only be identified in 50% of cases. Here, we describe and examine the genetic requirements for missing minor mitochondria (mmm), sterile affecting ciliogenesis (sac), and testes of unusual size (tous), three previously uncharacterized genes in Drosophila that are predicted to be components of the flagellar axoneme. Using Drosophila, we demonstrate that these genes are essential for male fertility and that loss of mmm, sac, or tous results in complete immotility of the sperm flagellum. Cytological examination uncovered additional roles for sac and tous during cytokinesis and transmission electron microscopy of developing spermatids in mmm, sac, and tous mutant animals revealed defects associated with mitochondria and the accessory microtubules required for the proper elongation of the mitochondria and flagella during ciliogenesis. This study highlights the complex interactions of cilia-related proteins within the cell body and advances our understanding of male infertility by uncovering novel mitochondrial defects during spermatogenesis.

Granulocyte Colony-Stimulating Factor Restored Impaired Spermatogenesis and Fertility in an AML-Chemotherapy Mice Model.

Leukemia and treatment of male patients with anticancer therapy (aggressive chemotherapy and/or radiotherapy) may lead to infertility or even permanent male sterility. Their mechanisms of spermatogenesis impairment and the decrease in male fertility are not yet clear. We showed that under acute myeloid leukemia (AML) conditions, alone and in combination with cytarabine (CYT), there was significant damage in the histology of seminiferous tubules, a significant increase in apoptotic cells of the seminiferous tubules, and a reduction in spermatogonial cells (SALL and PLZF) and in meiotic (CREM) and post-meiotic (ACROSIN) cells. In addition, we showed a significant impairment in sperm parameters and fertilization rates and offspring compared to control. Our results showed a significant decrease in the expression of glial cell line-derived neurotrophic factor (GDNF), macrophage colony-stimulating factor (MCSF) and stem cell factor (SCF) under AML conditions, but not under cytarabine treatment compared to control. In addition, our results showed a significant increase in the pro-inflammatory cytokine interleukin-1 (IL-1) alpha in whole testis homogenates in all treatment groups compared to the control. Increase in IL-1 beta level was shown under AML conditions. We identified for the first time the expression of GCSF receptor (GCSFR) in sperm cells. We showed that GCSF injection in combination with AML and cytarabine (AML + CYT + GCSF) extended the survival of mice for a week (from 6.5 weeks to 7.5 weeks) compared to (AML + CYT). Injection of GCSF to all treated groups (post hoc), showed a significant impact on mice testis weight, improved testis histology, decreased apoptosis and increased expression of pre-meiotic, meiotic and post- meiotic markers, improved sperm parameters, fertility capacity and number of offspring compared to the controls (without GCSF). GCSF significantly improved the spermatogonial niche expressed by increased the expression levels of testicular GDNF, SCF and MCSF growth factors in AML-treated mice and (AML + CYT)-treated mice compared to those groups without GCSF. Furthermore, GCSF decreased the expression levels of the pro-inflammatory cytokine IL-12, but increased the expression of IL-10 in the interstitial compartment compared to the relevant groups without GCSF. Our results show for the first time the capacity of post injection of GCSF into AML- and CYT-treated mice to improve the cellular and biomolecular mechanisms that lead to improve/restore spermatogenesis and male fertility. Thus, post injection of GCSF may assist in the development of future therapeutic strategies to preserve/restore male fertility in cancer patients, specifically in AML patients under chemotherapy treatments.

Dynamics of Spermatogenesis and Change in Testicular Morphology under 'Mating' and 'Non-Mating' Conditions in Medaka (Oryzias latipes).

Here, we report that the gross morphology of the testes changes under 'non-mating' or 'mating' conditions in medaka (Oryzias latipes). During these conditions, an efferent duct expands and a histological unit of spermatogenesis, the lobule, increases its number under 'non-mating' conditions. Based on BrdU labeling experiments, lower mitotic activity occurs in gonial cells under 'non-mating' conditions, which is consistent with the reduced number of germ cell cysts. Interestingly, the total number of type A spermatogonia was maintained, regardless of the mating conditions. In addition, the transition from mitosis to meiosis may have been retarded under the 'non-mating' conditions. The minimum time required for germ cells to become sperm, from the onset of commitment to spermatogenesis, was approximately 14 days in vivo. The time was not found to significantly differ between 'non-mating' and 'mating' conditions. The collective data suggest the presence of a mechanism wherein the homeostasis of spermatogenesis is altered in response to the mating conditions.

Expression of cell proliferation regulatory factors bricd5, tnfrsf21, cdk1 correlates with expression of clock gene cry1 in testes of Hu rams during puberty.

BACKGROUND: Cryptochrome 1 (cry1), the core regulator of the circadian clock, is essential for ontogeny and mammalian reproduction. Unlike in other tissues, the cry1 gene have noncircadian functions in spermatogenesis, which implies the unique role of cry1 gene in the development of testis. The role of cry1 during the puberty has not been described yet. This study aimed to explore the relationship between cry1 expression and spermatogenic cell numbers. METHODS AND RESULTS: We analyzed testicular tissues from Hu sheep aged 0-180 days by hematoxylin and eosin staining, measured cry1 and cell proliferation regulatory factors (bricd5, tnfrsf21, cdk1) expression by quantitative real-time PCR and characterized the transcription factor in the 5' flanking region of cry1 gene. The data revealed that the number of spermatocytes and early spermatocytes increased rapidly from 90 to 120 dpp (day postpartum). Correspondingly, there was a marked variation in the cry1 and cell proliferation related genes (bricd5, tnfrsf21, cdk1) mRNA expression in the testes from the age of 90 days to 180 days (p < 0.05). We also identified some transcription factors (tcfl5) related to cell proliferation. CONCLUSIONS: There is a significant causal relationship between the transcription level of cry1 gene in Hu sheep testes and the number of spermatogenic cells. It is speculated that cry1 gene may regulate the proliferation of spermatogenic cells by regulating the expression of cell proliferation related genes such as bricd5, tnfrsf21 and cdk1.