Testis Histology Research Articles

Men´s reproductive alterations caused by bisphenol A and its analogues: a review.

Male reproductive functions are an important area affecting men´s overall health and well-being. However, during the last years, there has been observed increasing incidence of male reproductive issues. The radical growth has been recorded parallelly with a massive expanse of industrialization and agricultural chemigation. Many groups of experts have begun to identify several potential factors and substances that may have adverse effects on men´s reproductive health. Since then, xenobiotics have become a major concern of many scientific studies. There is evidence that most of them have multigenerational and transgenerational effects on reproductive health, which is a serious problem for our population. Bisphenol A could be considered as one of the most studied endocrine disruptors. Until now, several negative effects of bisphenol A were associated with reduced weight testes, histological alterations, impairment spermatogenesis, and steroidogenesis as well as with testes or prostate cancer. Due to convincing evidence, bisphenol A has been started to replace by its analogues such as bisphenol B, S, F, in order to eliminate and suppress the risk of exposure to bisphenol A. However, it seems that a lack of toxicological analyses allows using of these hazardous substances in daily life. Their harmful effect was confirmed by the animal in vitro and in vivo models, while the epidemiological studies monitoring the impact of bisphenol analogues on men's reproductive health are markedly limited. This review provides information about the effects of bisphenol on reproductive health in men. At the same time, it is focused on physiological aspects of sperm viability, steroid hormone secretion, sperm motility, or testes histology in relation to bisphenols exposure.

Filgrastim (r-met-hu G-CSF) enhances the efficiency of spermatogenesis in prepubertal Bos indicus bulls.

This study aimed to test the effects of the drug r-met-hu-G-CSF (filgrastim) on spermatogenic efficiency in prepubertal Brahman bulls. Twelve intact, healthy prepubertal bulls were administered 0, 1 (LD=low dose) or 4 (HD=high dose) µg/Kg r-met-hu-G-CSF (daily for 4days), and haematological analysis was performed. Bulls were castrated (D0 or D60). BW (body weight) and SC (scrotal circumference) were recorded. Testis weight and volume were taken at castration with samples for testis histology and stereology: germ cell types, spermatids count and DSP (daily sperm production per gram)/g of testicular parenchyma. Testicular weight, volume, BW, SC and gonadosomatic index (GSI) were NS (LD-HD; p>.05). At D0 (age 11months), the most advanced germ cell types (maGCt) ranged from intermediate spermatogonia to pachytene spermatocytes. After 2months, control animals had round spermatids as maGCt, LD animals 75% round spermatids and 25% elongated spermatids, and HD animals round spermatids. Spermatids/testis were higher in LD (1.23±0.2 millions) than in controls (0.65±0.1 millions, p<.05). Spermatogenic efficiency (DSP/g) was higher in LD (5.4±0.4 million) than in controls (3.2±0.2 million, p<.01). In conclusion, r-met-hu-G-CSF raises spermatogenic efficiency in prepubertal Brahman bulls.

Hmga2 deficiency is associated with allometric growth retardation, infertility, and behavioral abnormalities in mice.

The high mobility group AT-hook 2 (HMGA2) protein works as an architectural regulator by binding AT-rich DNA sequences to induce conformational changes affecting transcription. Genomic deletions disrupting HMGA2 coding sequences and flanking noncoding sequences cause dwarfism in mice and rabbits. Here, CRISPR/Cas9 was used in mice to generate an Hmga2 null allele that specifically disrupts only the coding sequence. The loss of one or both alleles of Hmga2 resulted in reduced body size of 20% and 60%, respectively, compared to wild-type littermates as well as an allometric reduction in skull length in Hmga2−/− mice. Both male and female Hmga2−/− mice are infertile, whereas Hmga2+/− mice are fertile. Examination of reproductive tissues of Hmga2−/− males revealed a significantly reduced size of testis, epididymis, and seminal vesicle compared to controls, and 70% of knock-out males showed externalized penis, but no cryptorchidism was observed. Sperm analyses revealed severe oligospermia in mutant males and slightly decreased sperm viability, increased DNA damage but normal sperm chromatin compaction. Testis histology surprisingly revealed a normal seminiferous epithelium, despite the significant reduction in testis size. In addition, Hmga2−/− mice showed a significantly reduced exploratory behavior. In summary, the phenotypic effects in mouse using targeted mutagenesis confirmed that Hmga2 is affecting prenatal and postnatal growth regulation, male reproductive tissue development, and presents the first indication that Hmga2 function is required for normal mouse behavior. No specific effect, despite an allometric reduction, on craniofacial development was noted in contrast to previous reports of an altered craniofacial development in mice and rabbits carrying deletions of both coding and noncoding sequences at the 5′ part of Hmga2.

Defensive Impact of Co Q10 in Japanese Quail Males Raised Under Oxidative Stress Conditions

This study was investigated the protective role of (CoQ10) on reproductive dysfunction of males quail induced by hydrogen peroxide (H2O2). Forty Japanese quail male 45 days old were randomly allotted into four groups with ten replicate one per each , and treated for 28 days as follows : (G1): the first control group (G2): reserved standard ratio and (1%) H2O2 in drinking water. (G3): reserved production ratio + (100 mg Co Q10/kg diet) and (1%) H2O2 in drinking water. (G4): reserved production ratio + (100 mg Co Q10/kg diet) and normal water. The results showed that exposure of birds to H2O2 caused decrease in serum glutathione level (GSH), testosterone, luteinizing hormones (LH) and follicle stimulating hormone (FSH) hormones and this reflects on histology of testis by reducing seminiferous tubules diameter , area of germinal layer and germinal layer thickness. Whereas supplement of CoQ10 caused an increase the concentrations of theses, hormones in-group G4 as compared with group G2. It can noted that an addition was able to restore the oxidative stress birds (G3) to a state close to the natural state (G1). Though testicular histological modifications were made strides in grown-up Japanese quail male treated with CoQ10. In conclusion, the comes about of the display think about appeared that utilize of Co Q10 can easing the pernicious impacts on male regenerative work takingafter introduction toH2O, maybe through enhancement the antioxidant parameters or testicular capacities or other related endocrine organs.

TOXICOLOGICAL EVALUATION OF METHANOL SEED EXTRACT OF HUNTERIA UMBELLATA ON REPRODUCTIVE FUNCTIONS OF TREATED WISTAR RATS

Ethnomedicinal survey have shown that the different parts of Hunteria umbellata plant are used in Western and Southern parts of Nigeria in managing various human diseases such as sexually transmitted infections and to induce or augment labor. This is however, without information on its effect on reproductive functions. This study was therefore designed to ascertain if there is any health benefit or risk in the ingestion of Hunteria umbellata on reproductive functions. In this study, qualitative phytochemistry, acute toxicity test and the sub-chronic toxicity effects of 250, 500, and 1000 mg/kg/day of Hunteria umbellata (HU) methanol seed extract on reproductive functions in male and female Wistar rats were investigated for 90 days. To achieve this, effect of repeated doses of methanolic extracts of Hunteria umbellata seed were investigated on selected reproductive parameters of the test animals. Eighty (80) (40 male and 40 female) Wister rats, were randomly divided into 4 groups of 10 rats each. The first group received 10 ml/kg/day of distilled water and served as the control, while the second, third and fourth groups received 250, 500 and 1000 mg/kg/day of the extract for 90 days respectively. Blood samples were collected by retro-orbital puncture and delivered into plain tubes for hormonal assay, using ELISA hormone test kits (Biotec Laboratories Ltd, UK). After 90 days, the rats were anesthetized using chloroform. The histology of the testes and ovaries were also carried out. Result revealed that the prolonged treatment with methanol seed extracts of Hunteria umbellata for 90 days, caused minimal decreases (p&gt;0.05) in testosterone, estrogen, progesterone level in all the extract treated groups as compared with the control group. Also, no significant distortions were observed in the histology of testes and ovaries. The results suggests that the prolonged oral treatment with 250-1000 mg/kg/day of the methanol extract of the seed of Hunteria umbellata did not have any effect on the reproductive functions as well as the reproductive organs in males and females’ rats treated, and could be safe following repeated use.

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.

Effect of Oregostem® and imbalance diet on body performance and reproductive efficiency in male quails

This study was aimed to determine the influence of Oregostem® and imbalance diet on body performance, hematology and spermatogenesis of male quails. A total of 160 birds have been distributed into four groups (40 birds) each with 2 replicates starting at 12 weeks of age, the four treatments includes:(G1) was fed on balanced diet only. (G2) was received a balanced diet with Oregostem® with drinking water. While G3 was fed on imbalance diet (ground yellow corn) and G4 received imbalance diet with Oregostem®. The parameters including body performance, reproduction, blood and biochemical Figure and histology of testes were measured at 4th and 8th weeks from beginning of treatment. The results showed that the supplementation of Oregostem® caused a significant increase in body and right testis weight, gonadosomatic index, sperm count as well as live sperm in G2 relative to other groups at 4th and 8th weeks post treatment. In G3, WBC and lymphocytes decrease significantly with increasing hetrophils percentage and stress index at 4th and 8th weeks post treatment. Also there was a significant decrease of cholesterol with an increase of total protein and creatine kinase in G3 at 8th week of treatment. While the groups treated with Oregostem® did not differ from control group. This study concluded that liquid form of Oregostem® had a positive effect on body weight, sperm count with improvement hematological and biochemical parameters of the male quails.

Effects of glufosinate-ammonium on male reproductive health: Focus on epigenome and transcriptome in mouse sperm

Glufosinate-ammonium (GLA) is a widely used herbicide with emerging concern over its neural and reproductive toxicity. To uncover potential effects of GLA on male reproductive health in mammals, adult male C57BL/6J mice were administered 0.2 mg/kg·d GLA for 5 weeks. After examination on fertility, testis histology and semen quality in the GLA group, we performed deep sequencing to identify repressive epigenetic marks including DNA methylation and histone modifications (H3K27me3 and H3K9me3), together with mRNA transcript levels in sperm. Then, we integrated multi-omics sequencing data to comprehensively explore GLA-induced epigenetic and transcriptomic alterations. We found no significant difference either on fertility, testis histology or semen quality-related indicators. As for epigenome, the protein level of H3K27me3 was significantly increased in GLA sperm. Next generation sequencing showed alterations of these epigenetic marks and extensive transcription inhibition in sperm. These differential repressive marks were mainly distributed at intergenic regions and introns. According to results by Gene Ontology enrichment analysis, both differentially methylated and expressed genes were mainly enriched in pathways related to synapse organization. Subtle differences in genomic imprinting were also observed between the two groups. These results suggested that GLA predominantly impaired sperm epigenome and transcriptome in mice, with little effect on fertility, testis histology or semen quality. Further studies on human sperm using similar strategies need to be conducted for a better understanding of the male reproductive toxicity of GLA.

Dracaena arborea (Dracaenaceae) Increases Sexual Hormones and Sperm Parameters, Lowers Oxidative Stress, and Ameliorates Testicular Architecture in Rats with 3 Weeks of Experimental Varicocele.

Varicocele is a disease characterized by an abnormal dilation of the pampiniform plexus that drains the testis. The main objective of this work was to evaluate the curative effects of aqueous and ethanolic extracts of Dracaena arborea on some reproductive and antioxidant markers in rats with experimental varicocele. Following varicocele induction, rats (5 per group) were randomly partitioned into untreated varicocele, vitamin E-treated (150 mg/kg), aqueous extract-treated (500 mg/kg), and ethanolic extract-treated (100 mg/kg) animals. Two other groups served as normal and sham-operated. After 2 or 4 weeks of treatments, body and sex organ weights, spermatozoa characteristics, antioxidant status, NO level, sex hormones, and testis histology were measured. Animals with 3 weeks of varicocele showed a significant (p < 0.05–0.001) decrease in body and sex organ weights, total proteins, sperm characteristics, testosterone concentration, SOD, catalase, and total peroxidase activities. An increase in the plasmatic FSH, LH, and testicular MDA and NO concentrations was also recorded. Moreover, marked disorganization of the testicular architecture was observed. Treatment with D. arborea significantly reversed these impairments due to varicocele. For instance, after 4 weeks, treatment with aqueous extract of D. arborea significantly (p < 0.05–0.001) increased testes and epididymis weights, sperm viability (89.12 ± 1.09 vs 68.22 ± 1.42), sperm density (148.50 ± 2.59 vs 110.25 ± 2.51), and sperm motility (68.16 ± 2.39 vs 55.88 ± 3.20) in the left side, compared with varicocele-untreated rats. The extract also significantly (p < 0.05–0.001) decreased malondialdehyde level (2.19 ± 0.04 vs 3.50 ± 0.13) but elevated catalase (0.97 ± 0.03 vs 0.55 ± 0.03), SOD (0.5 ± 0.03 vs 0.15 ± 0.03), and peroxidase (65.80 ± 2.9 vs 40.95 ± 2.44) activities. Present results showed that D. arborea extracts possess antioxidant effects and improve sperm quality in male rats with an existing varicocele.

Changes in histology, protein expression, and autophagy in dairy goat testes during nonbreeding season†.

Seasonal reproduction contributes to increased chances of offspring survival in some animals. Dairy goats are seasonal breeding mammals. In this study, adult male Guanzhong dairy goats (10-12months old) were used. Testis size, semen quality, hormone level, apoptosis of germ cells, and autophagy of Sertoli cells were analyzed in dairy goats during the breeding (October) and nonbreeding (April) seasons. We found that, during the nonbreeding season for dairy goats, semen quality, follicle-stimulating hormone (FSH) levels, and testosterone levels were reduced, and the number of apoptotic germ cells increased. The proliferation with decrease activity of germ cells in dairy goat during the nonbreeding season was significantly affected. However, the testis size did not change seasonally. Interestingly, Sertoli cell autophagy was more active during the nonbreeding season. The expression levels of FSH receptor, wilms tumor 1, androgen binding protein, glial cell derived neurotrophic factor, and stem cell factor decreased in dairy goats during the nonbreeding season. In summary, our results indicate that spermatogenesis in dairy goats during the nonbreeding season was not completely arrested. In addition, germ cell apoptosis and the morphology of Sertoli cells considerably changed in dairy goats during the nonbreeding season. Sertoli cell autophagy is involved in the seasonal regulation of spermatogenesis in dairy goats. These findings provide key insights into the fertility and spermatogenesis of seasonal breeding animals.

P–002 Prediction model for salvage microdissection testicular sperm extraction in patients with failed conventional testicular sperm extraction

Abstract Study question Is there any intra-surgical parameter able to predict the outcome of salvage microdissection testicular sperm extraction (mTESE) in patients with previous failed TESE? Summary answer Among all the variables under consideration, only the seminiferous tubules (ST) caliber pattern found at high magnification was able to significantly predict the mTESE outcome... What is known already Several studies have demonstrated that no clinical or hormonal parameters are able to predict the outcome of a salvage mTESE performed in patients with previous sperm retrieval failure (SRF). It has been previously demonstrated that a prediction model with the combination of two intra-surgical parameters such as the STs caliber, defined as dilated tubule (DT), slightly dilated tubules (SDT) and not dilated tubules (NDT), and testis histology had an excellent discrimination ability (AUC 0.93) to distinguish between cases with and without the outcome, but such prediction model has not been tested in patients undergoing salvage mTESE. Study design, size, duration A prediction model was built on a dataset of 63 patients, 29–50 years old, undergoing unilateral (15 ) or bilateral (48) salvage mTESE after failed TESE from 2015 through 2019, with a resulting N = 111 testes under consideration. Two models were compared, one with STs and histology as covariates, the other with STs alone: the second model was chosen due to better discrimination... Participants/materials, setting, methods we assessed internal validity with a bootstrapping procedure for a realistic estimate of the performance of the prediction model in similar future patients with NOA undergoing salvage mTESE. We repeated the entire modeling process in 259 samples drawn with replacement from the original sample, and determined the performances (AUC, sensitivity, specificity) of the selected prediction model. Calibration (correspondence between the predicted and observed probabilities) was visually assessed by inspecting the calibration belt... Main results and the role of chance Sperm retrieval was successful in 24 out of 63 patients (38%): age, testis volume and hormonal parameters did not vary among patients with successful sperm retrieval (SSR) or SRF. The prevalent histological pattern was Sertoli cell only syndrome (69.6%), while hypospermatogenesis, maturation arrest and hyalinosis were found in 4.5%, 23% and 1.8% of cases. The STs pattern was heterogeneous, with DTs being found only in 23.4% of testes. Sperm were found in 69% of DTs, 29% of SDTs, and 5% of NDTs. The prediction model correctly classified 82.88% of patients and explained the 26.5% variability of the outcome. The STs pattern significantly predicted the mTESE outcome with a sensitivity of 62% and a specificity of 90.2%, PPV 69.2%, NPV 87%. Both SDT (OR 0.105, 95% CI 0.034–0.317, p &amp;lt; 0.0001) and NDT (OR 0.024, 95% CI 0.004–0.128, p &amp;lt; 0.0001) were negatively associated with the chance of retrieving sperm, the resulting prediction equation being Log (SSR)= 0.81 – 2.2 SDT – 3.7 NDT. The model had a clearly useful discrimination (AUC 0.813). The optimism corrected AUC was 0.7977, and the model was well calibrated (p = 1.00) with both the 80% and 95% calibration belts encompassing the bisector over the whole range of the predicted probabilities Limitations, reasons for caution The STs caliber pattern was subjectively evaluated at high magnification (24–36x) by comparing the individual ST appearance with the surrounding ones, however such evaluation was performed by the same experienced urologist with more than 1000 mTESE procedures performed to date. Wider implications of the findings: No clinical data but the STs appearance at high magnification could discriminate between patients with and without chances of SSR. These results reinforce the evidence supporting the superiority of mTESE compared to conventional TESE in retrieving sperm, particularly in lower prognosis patients with NOA such as those with previous SR. Trial registration number Not applicable

P–008 The diameter of standard seminiferous tubules varies with the different histological patterns in patients with non-obstructive azoosperm

Abstract Study question Does the standard, not-dilated seminiferous tubules (STs) diameter vary according with different testis histology patterns in patients with non-obstructive azoospermia (NOA)? Summary answer The standard STs diameter differed significantly among cases with different testis histology: hypospermatogenesis (HYPO) had the highest STs diameter compared to the other histological subgroups. What is known already During microdissection testicular sperm extraction (mTESE), the identification of dilated STs, as subjectively evaluated at high magnification by comparing their apparent caliber with that of the surroundings, is crucial to identify residual foci of intact spermatogenesis and to retrieve sperm. Previous studies have demonstrated that dilated STs contain sperm in most cases, but it is not clear why in some cases an apparent normal tubular caliber does not correspond to spermatogenesis integrity. Aim of the present study was to assess whether different histology patterns could affect the STs diameter. Study design, size, duration We retrospectively evaluated 168 patients with NOA undergoing unilateral (N = 91) or bilateral (N = 77) mTESE from 2018 through 2019. One or more biopsy samples representative of the overall appearance of the testicular parenchyma were taken from one (for unilateral mTESE) or both testes (for bilateral mTESE), was fixed in Bouin’s solution and sent to the pathologist. Histological analysis was conducted by the same experienced pathologist, who examined at least 100 different tubule sections per biopsy sample. Participants/materials, setting, methods Each tubule section (N = 100 per sample) was evaluated at 10X magnification with a micrometer to measure the tubule diameter, then the mean ST diameter was computed. The basal membrane (BM) thickening was evaluated in every section, and a score was assigned by multiplying the degree of BM fibrosis (mild=1, moderate=2, severe=3) for the number of sections (e.g. BM score for moderate fibrosis in 50 sections=2x50=100). Leydig cells hyperplasia (LCH), if present, was also annotated. Main results and the role of chance The median + interquartile range STs diameter was 140; 110–185 mm, while the median BM score was 100; 10–150. Sertoli cell only syndrome (SCO) was found in 51.1% of cases, focal SCO (FSCO) in 4,7%, early (EMA) and late (LMA) maturation arrest in 10.2 and 2.73% respectively, HYPO in 26.17% and hyalinosis (HL) in 5% of cases. LCH was found in 46.88% of samples. STs diameter, BM score and LCH differed significantly among the different histological patterns: STs diameter was 125; 100–148 in SCO, 162; 102–187 in FSCO, 130; 100–175 in EMA, 145; 130–195 in LMA, 205; 170–240 in HYPO and 57.5; 42.5–100 in HL. HYPO samples also had the lowest BM score (20;1–100; p &amp;lt; 0.0001) and LCH prevalence (23.8%, p &amp;lt; 0.0001) compared to the other histological subgroups (HS). A multinomial logistic regression for prediction of different histological subgroups was run with STs diameter, BM score and LCH as candidate predictors: the model explained the 29% of variability of the outcome and correctly classified 69% of cases. STs diameter significantly predicted FSCO (RR 1.02, 95% CI 1.0–1.04), LMA (RR 1.02, 95% CI1–1.04) and HYPO (1.03, 95%CI 1.02–1.04), while BM score significantly predicted HL (RR 1.07, 95%CI 1.02–1.13). Limitations, reasons for caution The STs were carefully cut before extraction in order to preserve their structural integrity, however the accuracy of such a method of estimating the STs diameter needs to be assessed by further studies. Wider implications of the findings: The identification of dilated STs remains the best strategy to retrieve sperm by mTESE, since larger STs diameters are mostly associated with the more favorable histological patterns. However, dilated STs may be also found in cases with LMA, which explains why in some cases dilated STs do not contain sper. Trial registration number Not applicable

CRISPR/Cas9-based genetic screen of SCNT-reprogramming resistant genes identifies critical genes for male germ cell development in mice

Male germ cells undergo complex developmental processes eventually producing spermatozoa through spermatogenesis, although the molecular mechanisms remain largely elusive. We have previously identified somatic cell nuclear transfer-reprogramming resistant genes (SRRGs) that are highly enriched for genes essential for spermatogenesis, although many of them remain uncharacterized in knockout (KO) mice. Here, we performed a CRISPR-based genetic screen using C57BL/6N mice for five uncharacterized SRRGs (Cox8c, Cox7b2, Tuba3a/3b, Faiml, and Gm773), together with meiosis essential gene Majin as a control. RT-qPCR analysis of mouse adult tissues revealed that the five selected SRRGs were exclusively expressed in testis. Analysis of single-cell RNA-seq datasets of adult testis revealed stage-specific expression (pre-, mid-, or post-meiotic expression) in testicular germ cells. Examination of testis morphology, histology, and sperm functions in CRISPR-injected KO adult males revealed that Cox7b2, Gm773, and Tuba3a/3b are required for the production of normal spermatozoa. Specifically, Cox7b2 KO mice produced poorly motile infertile spermatozoa, Gm773 KO mice produced motile spermatozoa with limited zona penetration abilities, and Tuba3a/3b KO mice completely lost germ cells at the early postnatal stages. Our genetic screen focusing on SRRGs efficiently identified critical genes for male germ cell development in mice, which also provides insights into human reproductive medicine.

Intrasurgical parameters associated with successful sperm retrieval in patients with non‐obstructive azoospermia undergoing salvage microdissection testicular sperm extraction

Patients with non-obstructive azoospermia with a previously failed conventional testicular sperm extraction may undergo a salvage microdissection testicular sperm extraction with the probability of successful sperm retrieval being almost dependent upon the number of previous surgical attempts and to different histopathologic categories. To determine whether the seminiferous tubules pattern and the histological categories could affect the sperm retrieval rate in patients with non-obstructive azoospermia undergoing salvage microdissection testicular sperm extraction after failed conventional testicular sperm extraction. Seventy-nine patients undergoing unilateral or bilateral salvage microdissection testicular sperm extraction were evaluated. During microdissection testicular sperm extraction, if present, dilated tubules were retrieved, otherwise, tubules with slightly larger caliber than that of the surroundings were removed. When no dilated tubule or tubule with slightly larger caliber was found, not dilated tubules were excised. A prediction model was built with seminiferous tubules pattern and testis histology as covariates. Sperm retrieval was successful in 30 out of 79 patients. The prediction model correctly classified 88.3% of cases, explained the 29.7% variability of the outcome, and significantly predicted the microdissection testicular sperm extraction outcome with a sensitivity of 67.7% and a specificity of 90.2%, Both tubules with slightly larger caliber and not dilated tubules were negatively associated with the chance of retrieving spermatozoa. Among the histological categories, only early maturation arrest was significant to the model (log(SSR)=0.57 - 1.9SDT - 3.3NDT - 1.76EMA) (where SSR is sperm retrieval rate, SDT is tubule with slightly larger caliber, NDT is not dilated tubule, and EMA is early maturation arrest). The model had a clearly useful discrimination (area under the curve = 0.814), the estimated performance was 0.8105, and internal calibration was acceptable (p>0.05). Seminiferous tubules pattern and testis histology may reliably explain the salvage microdissection testicular sperm extraction outcome in all patients with non-obstructive azoospermia apart from those with early maturation arrest, where the homogeneous apparent seminiferous tubules pattern may be misleading. The outcome of salvage microdissection testicular sperm extraction can be predicted by the same intrasurgical parameters that have been demonstrated to predict the outcome of microdissection testicular sperm extraction in naïve patients with non-obstructive azoospermia.

Dosing-time dependent testicular toxicity of everolimus in mice

The circadian timing system controls many biological functions in mammals including drug metabolism and detoxification, cell cycle events, and thus may affect pharmacokinetics, target organ toxicity and efficacy of medicines. Selective mTOR (mammalian target of rapamycin) inhibitor everolimus is an immunosuppressant and anticancer drug that is effective against several cancers. The aim of this study was to investigate dosing-time dependent testicular toxicity of subacute everolimus administration in mice. C57BL/6 J male mice were synchronized with Light-Dark (12h:12 h) cycle, with Light-onset at Zeitgeber Time (ZT)-0. Everolimus (5 mg/kg/day) was administered orally to mice at ZT1rest-span or ZT13activity-span for 4 weeks. Body weight loss, clinical signs, changes in testicular weights, testis histology, spermatogenesis and proliferative activity of germinal epithelium of seminiferous tubules were examined. Steady-state everolimus concentrations in testes were determined with validated HPLC method. Everolimus toxicity was less severe following dosing at ZT13 compared to ZT1, as shown with least body weight loss (p<0.001), least reductions in testes weights (p<0.001) and least histopathological findings. Everolimus-induced histological changes on testes included vacuolisation and atrophy of germinal epithelium, and loss of germinal cell attachment. The severity of everolimus-induced histological toxicity on testes was significantly more evident in mice treated at ZT1 than ZT13 (p<0.001). Spermatogenic cell population significantly decreased when everolimus administered at ZT1 compared to ZT13 (p<0.001). Proliferative activity of germinal epithelium was significantly decreased due to treatment at ZT1 compared to ZT13 (p<0.001). Everolimus concentrations in testes indicated a pronounced circadian variation, which was greater in mice treated at ZT1 compared to ZT13 (p<0.05). Our study revealed dosing-time dependent testicular toxicity of everolimus in mice, which was greater in severity when everolimus administered at early rest-span (daytime-ZT1) than early activity-span (nighttime-ZT13). These findings support the concept of everolimus chronotherapy for minimizing reproductive toxicity and increasing the tolerability of everolimus, as a clinical advantage.

Paternal MTHFR deficiency leads to hypomethylation of young retrotransposons and reproductive decline across two successive generations.

ABSTRACT5,10-Methylenetetrahydrofolate reductase (MTHFR) is a crucial enzyme in the folate metabolic pathway with a key role in generating methyl groups. As MTHFR deficiency impacts male fertility and sperm DNA methylation, there is the potential for epimutations to be passed to the next generation. Here, we assessed whether the impact of MTHFR deficiency on testis morphology and sperm DNA methylation is exacerbated across generations in mouse. Although MTHFR deficiency in F1 fathers has only minor effects on sperm counts and testis weights and histology, F2 generation sons show further deterioration in reproductive parameters. Extensive loss of DNA methylation is observed in both F1 and F2 sperm, with >80% of sites shared between generations, suggestive of regions consistently susceptible to MTHFR deficiency. These regions are generally methylated during late embryonic germ cell development and are enriched in young retrotransposons. As retrotransposons are resistant to reprogramming of DNA methylation in embryonic germ cells, their hypomethylated state in the sperm of F1 males could contribute to the worsening reproductive phenotype observed in F2 MTHFR-deficient males, compatible with the intergenerational passage of epimutations.

Effect of nickel chloride on reproductive parameters and testicular oxidative stress biomarkers in male guinea pigs

The soil and agricultural products pollution by nickel represents an important public health risk in agrarian areas such as the Dschang in Cameroon. This study was designed to evaluate the reproductive toxicity and oxidative stress potential of nickel chloride in male guinea pig. Four groups of adult male guinea pigs were orally treated with nickel chloride at doses of 0, 17.50, 26.25 and 52.50 mg/kg bw for 90 days. At the end of the experimental period, all animals were sacrificed, and blood samples and vital organs were collected for different analysis. Treatment of male guinea pigs with 52.50 mg/kg nickel chloride resulted in increased kidney weight and volume and decreased weights of the sex accessory glands (seminal vesicle + prostate + coagulating glands), epididymis and vas deferens. The 52.50 mg/kg dose of nickel chloride decreased (p&lt;0.05) the animal’s sperm mobility, number and viability, while it increased (p&lt;0.05) sperm micro and macrocephalies. Assessment of biochemical parameters of toxicity revealed increase (p&lt;0.05) of serum creatinine and aminotransferases activities in the nickel chloride-exposed guinea pigs (52.50 mg/kg). The nickel chloride (52.50 mg/kg) also promoted oxidative stress, through decrease (p&lt;0.05) of superoxide dismutase and catalase activities, as well as increase (p&lt;0.05) in lipid peroxydation. In addition, histology of testis revealed disrupted germ cell arrangement, decreased concentration of sperms in the lumen of the seminiferous tubules and degraded germinal epithelium in the animals exposed to nickel chloride. In conclusion, results obtained in this study revealed that nickel chloride perturbs male reproductive system and induced oxidative stress.

Reproduction Control in Coptodon zillii using Abrus precatorius Root Bark Diets as Fertility Inhibitor Agent

The antifertility effects of Abrus precatorius root bark meal (APRB) on Coptodon zillii juvenile was determined by a 42 days feeding experiment, with the inclusion levels of 0g, 6g, 12g, 18g and 24g/kg diet. Water volume of 20 litres in a plastic tank (50cm x 27cm x 35 cm dimension) was maintained throughout the period of experiment. Coptodon zillii was fed at 4% body weight. In male fish, testis weight, milt volume, milt count and motility duration reduced with increase in inclusion level and there were significant differences (P&lt;0.05) in the treatments used. Histology of testis at 0g of APRB/kg diet showed primary and secondary spermatocytes in the seminiferous tubule, connective tissue was clearly seen. In treatments 6g, 12g, 18g and 24g APRB/kg diets, there were increase in interstitial cell, eroded connective tissue and necrosis. In female fish, ovary weight, fecundity, relative fecundity and gonadosomatic index reduced with increase in the concentration of treatments used. Histology of the ovary at 0g APRB/kg diet showed developing oocytes while treatments 6g, 12g, 18g and 24g revealed reduced oocytes, increase in interstitial cell, alteration in ovary development and necrosis. The research revealed that Abrus precatorius root bark meal may be effective in controlling reproduction in Coptodon zillii.

Inactivation of testis-specific gene C4orf46 is dispensable for spermatogenesis and fertility in mouse.

Several genes have been reported to be involved in spermatogenesis but their functional importance in male fertility is yet needed to be elucidated. Therefore, in current research, we focused to explore the in vivo role of evolutionary conserved and testis-specifically expressed, C4orf46, gene in male mouse fertility and spermatogenesis. The expression profile of C4orf46 is specific to testes and expressed in testes from 7 days of postpartum to onward.Thus, we generated the C4orf46 knockout mice by utilizing CRISPR/Cas9 genome editing technology and examined gene function in spermatogenesis and fertility. Surprisingly, C4orf46 knockout mice were completely fertile, displayed normal testes morphology, however, higher sperm contents were observed in knockout mice compared to wild type (WT) littermates. Subsequently, intact testis histology and architecture of seminiferous tubules were observed in C4orf46 knockout and WT mice. Similarly, sperm morphology and swimming velocity of C4orf46 knockout mice were comparable with the WT littermates. Furthermore, all type of germ cells ranging from spermatogonia to mature spermatozoa were observed in the testes and epididymis sections of C4orf46 knockout mice suggesting that disruption of C4orf46 did not impact spermatogenesis. Moreover, meiotic prophase I progression was normal, and each type of cell population was comparable between knockout and WT mice. Overall, finding from this research indicates that C4orf46 is not an essential gene for fertility in mice. This study will help researchers to avoid the repetition and duplication of efforts, and to explore the genes that are indispensable for spermatogenesis and male fertility.

Marsilea crenata ethanol extract prevents monosodium glutamate adverse effects on the serum levels of reproductive hormones, sperm quality, and testis histology in male rats

Background and Aim:Marsilea crenata is an aquatic plant that contains high antioxidants level and could prevent cell damages caused by free radicals. The present study aimed to investigate the effect of M. crenata ethanol extract on luteinizing hormone (LH), testosterone levels, sperm quality, and testis histology of adult male rats induced by monosodium glutamate (MSG).Materials and Methods:This study randomly divided 48 male rats into eight groups (n=6): control group; MSG group (4 mg/g body weight [b.w.] for 30 days); MS1, MS2, and MS3 groups (4 mg/g b.w. MSG and M. crenata ethanol extract at dose 0.216, 0.432, and 0.648 mg/g b.w., respectively, for 30 days); and S1, S2, and S3 groups (M. crenata ethanol extract at dose of 0.216, 0.432, and 0.648 mg/g b.w., respectively, for 30 days). The blood sample was collected on days 0 and 30 to determine the LH and testosterone levels. The animals were dissected on day 30, and the testes were isolated for morphometric, histology (spermatogenic cell number), and malondialdehyde (MDA) examination. Moreover, semen was collected to determine the sperm quality parameter.Results:The LH and testosterone levels significantly increased (p<0.05) after M. crenata administration at all doses. The higher dose of M. crenata ethanol extract demonstrated a high decrease in MDA level in MSG-treated rat testis; increase of spermatogonia, spermatocytes, spermatids, and Leydig cells number; and increase of seminiferous tubular diameter and germinal epithelium thickness.Conclusion:The ethanol extract of M. crenata can improve the levels of LH, testosterone, sperm quality, number of testis morphometric, spermatogenic, and Leydig cells in MSG-treated male rats.