Seminiferous Tubules Of Testis Research Articles

CCDC189 depletion leads to oligo-astheno-teratozoospermia and male infertility in mice†.

In male reproductive system, proteins containing the coiled-coil domain (CCDC) are predominantly expressed in specific regions including the testis, epididymis, seminal vesicle, and prostate. They play a vital role in centriole formation, sperm motility and flagellar development in male gametes. Despite being highly expressed in the testis, the exact physiological function of the coiled-coil domain-containing 189 (Ccdc189) gene remain largely unclear. Our research provides a comprehensive and detailed investigation into the localization of CCDC189 protein within the testis seminiferous tubules. CCDC189 specifically expressed in spermatocytes, round spermatids, and elongating spermatids in mouse testis. The deletion of Ccdc189 in mouse leads to male infertility, characterized by significantly reduced sperm counts and motility. Abnormally shaped spermatozoa with irregular tails, exhibiting shortened and twisted morphology, were observed in the seminiferous tubules. Electron microscopy revealed disordered and missing peripheral microtubule doublets (MTD) and outer dense fibers (ODF) in the sperm flagella, accompanied by a consistent absence of central pairs (CP). The knockout of Ccdc189 resulted in oligo-astheno-teratozoospermia, which is characterized by low sperm count and reduced sperm motility and abnormal morphology. Furthermore, we identified poly(A)-binding protein cytoplasmic 1 (PABPC1) and PABPC2 as interacting proteins with CCDC189. These proteins belong to the PABP family and are involved in regulating mRNA translational activity in spermatogenic cells by specifically binding to poly(A) tails at the 3' ends of mRNAs.

The Protective effects of Vitamin E against alterations of rat testis structure induced by deltamethrin; histological, ultrastructure, and biochemical study

ABSTRACT Deltamethrin is a widely used synthetic pyrethroid pesticide. It causes reproductive toxicity. Aim of the work: it evaluates the impact of vitamin E in restoration of the testicular integrity of albino rats after toxicity induced by Deltamethrin. Thirty-six adult male albino rats were included, and they were further sub-divided into four experimental groups; Group A: six rats served as controls. Group B (Model): 10 rats equally divided into two sub-groups (B1): the rats received deltamethrin dissolved in oil in a dose of 0.6 mg/kg/daily by nasogastric gavage for 2 weeks. (B2): the rats received Deltamethrin in the same dose of group B1 for 1 month. Group C (Protected): 10 rats equally divided into two sub-groups (C1): the rats received deltamethrin orally 0.6 mg/kg/day concomitant with Vitamin E dissolved in 1 ml of corn oil in a dose 200 mg/kg/day by nasogastric gavage for 2 weeks. (C2): the rats received deltamethrin concomitant with Vitamin E in the same dose of group C1 for 1 month. Group D (Treatment): 10 rats received deltamethrin for 1 month followed by Vitamin E for another month in the same previously prescribed doses. Significant decreases in serum testosterone level, GSH, catalase activity, and significant increase in MDA in the deltamethrin-treated group were detected. Moreover, histological and ultrastructural examinations of the testis seminiferous tubules showed detrimental alterations in the deltamethrin group which were duration dependent. Vitamin E administration reversed such alterations. Vitamin E ameliorates the testicular dysfunction caused by Deltamethrin.

A new insight of cadmium-induced cellular evidence of autophagic-associated spermiophagy during spermatogenesis.

Autophagy plays a dynamic role in spermatozoa development during spermatogenesis. However, the disruption of autophagic flux induces cell death under metal toxicity and severe oxidative stress. Therefore, we hypothesized that cadmium-induced autophagy might be involved in this mechanism. To verify this hypothesis, we studied cadmium-induced cellular evidence of autophagic-associated spermiophagy within the testis. In the present study, treatment with cadmium caused nuclear depressive disorders and vacuolated mitochondrial damage of Sertoli cells. In addition, spermiophagy through the cellular evidence of spermatozoa phagocytosis, the high lysosomal activity (lysosome engulfment and phagolysosome), and autophagy activity (autolysosome and autophagosome) were observed in the Sertoli cells. The immunohistochemistry of lysosomal membrane protein (LAMP2) to target the phagocytosis of spermatozoa revealed that the immunoreactivity of LAMP2 was overstimulated in the luminal compartment of testis's seminiferous tubules. In addition, the immunohistochemistry and immunofluorescence of autophagy-related protein and microtubule-associated light chain (LC3) results showed the strong immunoreactivity and immunosignaling of LC3 in the Sertoli cells of the testis. Moreover, cadmium caused the overactivation of the expression level of autophagy-related proteins, autophagy-related gene (ATG7), (ATG5), beclin1, LC3, sequestosome 1 (P62), and LAMP2 which were confirmed by western blotting. In summary, this study demonstrated that hazards related to cadmium-induced autophagic-associated spermiophagy with the disruption of autophagic flux, providing new insights into the toxicity of cadmium in mammals and representing a high risk to male fertility.

P-143 Will Use of Testicular sperm in ICSI cycles increase Embryo Aneuploidy?

Abstract Study question Will use of surgically retrieved Testicular Sperms (TESA) in Intra-Cytoplasmic Sperm Injection (ICSI) cycles increase embryo aneuploidy? Summary answer Use of TESA sperms for ICSI seems safe and doesn’t increase embryo aneuploidy. TESA performed for various indications also showed no variation with embryo aneuploidy. What is known already Invention of ICSI and improved surgical sperm retrieval techniques have helped men with severe male factor infertility to father their own genetic child. Sperms retrieved from seminiferous tubules of testes are considered immature as they haven’t completed the process of spermiogensis. It is hypothesized that use of TESA sperm for ICSI can increase embryo aneuploidy, which is still uncertain. Study design, size, duration This retrospective study conducted at a private fertility clinic from 2014–2022 (n = 265 patients; total of 860 embryos evaluated). Study populationTESA sperm used and PGT-A done (n = 66). TESA group was further sub-divided into 2 groups based on the indication for TESA,Non-Obstructive Azoospermia(n = 26) and Raised SDF(n = 40). Control Population Ejaculated sperm used and PGT-A done (n = 199) Only younger women(<37yrs) with self-gametes and RIFconsidered in this study. RIF was defined as women with 2 failed IVFcycles in thepast with at least 4 blastocystsbeing transferred. Participants/materials, setting, methods All fertilized oocytes inseminated by ICSI and subjected to extended blastocyst culture. TESA done as per our clinic’s SOP and for control group sperm preparation done by density gradient method. Trophectoderm biopsy was performed and sent for PGT-A to the genetic lab. Next-Generation sequencing (NGS) was the Comprehensive Chromosomal technique (CCS) used for assessing embryo ploidy status. Aneuploidy across all groups compared. Main results and the role of chance Following were the Aneuploidy rates of TESA sperm, Ejaculate sperm group and TESA done for NOA and SDF respectively. Ejaculate Sperm – Aneuploidy%- 42% TESA Sperm (irrespective of the indication)- Aneuploidy%- 44% (p = 0.6260) TESA Sperm (done for NOA)- Aneuploidy%- 44% (p = 0.7504) TESA Sperm (done for raised SDF) – Aneuploidy%- 32% (p = 0.05) Embryo aneuploidy was comparable between Ejaculate and TESA sperm group. Though TESA sperm is considered immature, it didn’t seem to increase embryo aneuploidy. Embryo Aneuploidy from TESA sperm ICSI cycles done for NOA or raised SDF also showed no altering trend. Data from this study is suggestive that TESA sperm doesn’t alter embryo ploidy status. Limitations, reasons for caution Data is retrospective with small sample size and unequal distribution. A well designed Randomized Trial might help test the inference of this study further. Wider implications of the findings Incidence of male infertility and use of ICSI is on a rise globally. Interventions to optimize embryo implantation and reproductive outcomes will help with better success rates and shorten duration to conception. Further research is warranted with severe male factor infertility to improve sperm selection techniques and reproductive outcomes. Trial registration number not applicable

Comparative transcriptome analysis identified crucial genes and pathways affecting sperm motility in the reproductive tract of drakes with different libido

Libido can affect the semen quality of male, and the sperm motility in semen quality parameters is a reliable index to evaluate the fertility of male. In drakes, the sperm motility is gradually acquired in testis, epididymis, and spermaduct. However, the relationship between libido and sperm motility in drakes has not been reported and the mechanisms of testis, epididymis, and spermaduct regulating the sperm motility of drakes are unclear. Therefore, the purpose of the present study was to compare the semen quality of drakes with libido level 4 (LL4) and libido level 5 (LL5), and tried to identify the mechanisms regulating the sperm motility in drakes by performing RNA-seq in testis, epididymis, and spermaduct. Phenotypically, the sperm motility of drakes (P < 0.01), weight of testis (P < 0.05), and organ index of epididymis (P < 0.05) in the LL5 group were significantly better than those in LL4 group. Moreover, compared with the LL4 group, the ductal square of seminiferous tubule (ST) in testis was significantly bigger in the LL5 group (P < 0.05), and the seminiferous epithelial thickness (P < 0.01) of ST in testis and lumenal diameter (P < 0.05) of ductuli conjugentes/dutus epididymidis in epididymis were significantly longer in the LL5 group. In transcriptional regulation, in addition to KEGG pathways related to metabolism and oxidative phosphorylation, lots of KEGG pathways associated with immunity, proliferation, and signaling were also significantly enriched in testis, epididymis, and spermaduct, respectively. Furthermore, through the integrated analysis of coexpression network and protein-protein interaction network, 3 genes (including COL11A1, COL14A1, and C3AR1) involved in protein digestion and absorption pathway and Staphylococcus aureus infection pathway were identified in testis, 2 genes (including BUB1B and ESPL1) involved in cell cycle pathway were identified in epididymis, and 13 genes (including DNAH1, DNAH3, DNAH7, DNAH10, DNAH12, DNAI1, DNAI2, DNALI1, NTF3, ITGA1, TLR2, RELN, and PAK1) involved in Huntington disease pathway and PI3K-Akt signaling pathway were identified in spermaduct. These genes could play crucial roles in the sperm motility of drakes with different libido, and all data the present study obtained will provide new insights into the molecular mechanisms regulating sperm motility of drakes.

Effects of Insulin-degrading enzyme on the proliferation of swine Sertoli cells.

Sertoli cells, the only somatic cells in testis seminiferous tubules, provide a supporting microenvironment for male germ cells and play essential roles in spermatogenesis. The insulin-degrading enzyme (IDE), a ubiquitous zinc peptidase of the inverzincin family, plays crucial role in sperm production, as IDE-knockout mice presented decreased testis weight and impaired sperm viability and morphology. However, whether and how IDE affects swine Sertoli cell proliferation remains unclear. Thus, in the present study, we aimed to evaluate the effects of IDE on the proliferation of swine Sertoli cells, as well as its underlying molecular mechanism. After knocking down IDE expression with small interfering RNA transfection, we analyzed the proliferation of swine Sertoli cells as well as the expression of related regulatory factors (WT1, ERK, and AKT). The results showed that IDE knockdown promoted swine Sertoli cell proliferation and increased WT1 expression, possibly through activating ERK and AKT. Overall, our findings suggest that IDE may be involved in male reproduction by regulating Sertoli cell proliferation, which provides new information to better understand the regulatory mechanisms of swine Sertoli cells and improve the reproductive traits of male pigs.

Adverse effects of pristine and aged polystyrene microplastics in mice and their Nrf2-mediated defense mechanisms with tissue specificity.

Health hazards associated with microplastics (MPs) remain largely unknown, and the effects of aged MPs, one of their persistent forms, are poorly characterized. Male ICR mice were intratracheally instilled with 0.01 and 1mg/day pristine and ultraviolet (UV)-aged polystyrene microplastics (PS and APS) with an average diameter of 4 - 5μm daily for 1week. UV irradiation caused the PS to have a rough surface, become fragmented, and increase their carbonyl groups. Both PS and APS caused structural damage to the mouse gut, liver, spleen, and testis. Inflammatory infiltration in liver, swollen and congested gut, and loose spleen globules, as well as the loose interstitium of the seminiferous tubules in testis were found in 1mg/day APS group. Increases in serum alanine aminotransferase and immunoglobulin A levels in 1mg/day APS group (p < 0.05) demonstrated that APS exposure could induce greater liver and spleen functional damage than PS. Meanwhile, triglyceride and total cholesterol levels in liver were enhanced in 1mg/day APS group (p < 0.05). Superoxide dismutase and glutathione contents in 0.01 and 1mg/day APS groups significantly decreased (p < 0.05), which suggesting that PS and APS could interfere with the antioxidant capacity in mice. Nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) levels in the PS and APS groups showed significant increases in the liver and testis (p < 0.05), and a significant decrease in the spleen (p < 0.05), which were analyzed to get a first survey for Nrf2/HO-1-mediated tissue-specific defense mechanisms. In conclusion, acute exposure to PS and APS induced potential metabolic disorders, and APS could produce more serious immune damage and reproductive toxicity. These findings provide new insights in health risk assessment of aged MPs.

Retrospective detection of monkeypox virus in the testes of nonhuman primate survivors.

Close contact through sexual activity has been associated with the spread of monkeypox virus (MPXV) in the ongoing, global 2022 epidemic. However, it remains unclear whether MPXV replicates in the testes or is transmitted via semen to produce an active infection. We carried out a retrospective analysis of MPXV-infected crab-eating macaque archival tissue samples from acute and convalescent phases of infection of clade I or clade II MPXV using immunostaining and RNA in situ hybridization. We detected MPXV in interstitial cells and seminiferous tubules of testes as well as epididymal lumina, which are the sites of sperm production and maturation. We also detected inflammation and necrosis during the acute phase of the disease by histological analysis. Finally, we found that MPXV was cleared from most organs during convalescence, including healed skin lesions, but could be detected for up to 37 d post-exposure in the testes of convalescent macaques. Our findings highlight the potential for sexual transmission of MPXV in humans.

Detection of the ADAM proteins in the ejaculated bovine sperm

The current study aimed to detect specific types of ADAM proteins in the bovine sperm. Histology study of tissue of male reproduction was involved with testis, head, body, and the tail of the epididymis. Results showed that the testis composed of the parenchyma of testis contained the seminiferous tubules which are surrounded by two layers externally tunica vaginalis and internally tunica albuginea. PAS was positive in the seminiferous tubules of testis and lumen, epithelium of the head, and body of epididymis, Whereas Alcian blue was very low intensity stains with sections and contents of the head, body and tail of the epididymis. Fresh ejaculated bovine sperms were collected and separated from seminal plasma, and proteins of sperm were extracted and run on an electrophoresis gel. Then, the bands of proteins have been stained with coomassie stain, after that, the pieces of proteins were cut and separated from gels and amino acids were extracted after the digestion of proteins, and uploaded to a mass spectrometry machine. Our data was profiled and analysed and detected characterised ADAM proteins included: ADAM1, ADAM2, ADAM3A, ADAM10 and ADAM32, and uncharacterized ADAM proteins included: ADAM1B, ADAM7, ADAM9, ADAM12, ADAM15, ADAM17.

Two complementary approaches for efficient isolation of Sertoli cells for transcriptomic analysis.

Sertoli cells (SCs) are the only somatic cells that reside in seminiferous tubules of testis. They directly interact with and support the development of germ cells, thus have an indispensable role in the process of spermatogenesis. SCs first appear in a proliferative state and then, with the initiation of the first wave of spermatogenesis, progress to a mature “nurturing” state which supports lifelong continuous sperm production. During this development, the SC transcriptome must adapt rapidly as obstacles in SC maturation often result in deficiencies in male fertility. Due to its importance in spermatogenesis, a reliable, rapid, and precise method for the isolation of high purity, viable and unadulterated SC has been largely missing. We have developed an improved method for the preparation of a testicular single cell suspension comprised of two alternative protocols to separate SCs from the rest of the testicular cells by FACS. The first sorting scheme is based on their co-expression of surface specific markers, FSHr and Occludin-1, while the second focuses on the co-staining of SCs with FSHr-specific antibody and Hoechst 33342, which discriminates DNA content of testicular cells. The entire procedure can be completed in less than 3 h which permits the analysis of the development-related transcriptional profile of these cells. Notably, our comparative study showed that this method resulted in a SC transcriptome that is largely comparable to SCs which were briskly isolated due to their cell-specific expression of fluorescent protein. Interestingly, we also show that SCs sorted as FSHr+Occludin+ cells contained a tangible portion of transcripts from all types of testicular germ cells. Sorting of SCs according to their 2C DNA content significantly reduced the presence of these transcripts, thus seems to be the most suitable approach for accurate determination of the SC transcriptome. We believe that these novel approaches for the isolation of SCs will assist researchers in the elucidation of their function as well as their role in spermatogenesis and disorders related to male infertility.

Xenotransplantation of Human Spermatogonia Into Various Mouse Recipient Models.

Spermatogonial stem cells are the foundation of continuous spermatogenesis in adult mammals. Xenograft models have been established to define human SSCs, mostly using infertile and immune-deficient mice as the recipients for human germ cell transplantation. However, it is time-consuming to prepare such recipients using irradiation or chemotherapeutic agents, and this approach may also introduce confounding factors when residual endogenous germ cells recover in transplanted recipients. It remains to be determined whether immune-competent genetically infertile mice can be suitable recipients for xenotransplantation. In this study, we observed similar engraftment efficiencies when using spermatogonia from human biopsied testes across immune-deficient nude mice, immune-competent ICR mice, and genetically infertile Kit w/w-v mice, suggesting minimal immunological rejection from immune-competent mouse recipients upon xenotransplantation of human germ cells. More importantly, we derived EpCAM negative and TNAP positive spermatogonia-like cells (SLCs) from human pluripotent stem cells (PSCs), which highly expressed spermatogonial markers including PLZF, INTERGRINα6, TKTL1, CD90, and DRMT3. We found that upon transplantation, these SLCs proliferated and colonized at the basal membrane of seminiferous tubules in testes of both immune-deficient nude mice and Kit w/w-v mice, though complete spermatogenesis would likely require supporting human signaling factors and microenvironment. Taken together, our study functionally defined the cell identity of PSC-derived SLCs, and supported xenotransplantation using genetically infertile recipients as a convenient model for functionally evaluating spermatogonia derived from different species.

Administration of olaquindox impairs spermatogenesis and sperm quality by increasing oxidative stress and early apoptosis in mice

Olaquindox (OLA), a potent antibacterial agent, has been widely used as a feed additive and growth promoter in animal husbandry. Our previous study has shown that OLA administration in female mice could markedly cause sub-fertility. Here we established the model in male mice to investigate the toxic effects of OLA on mammalian spermatozoa quality and fetal development. After continuous 45 days of OLA gavage, the dosage of 60 mg/kg/day (high dose) significantly affected body weight, organ weights and coefficients, and the morphology of the testis seminiferous tubule in male mice. Dosage of 60 mg/kg/day also reduced sperm count, motility, and viability. OLA at both low-dose (5 mg/kg/day) and high-dose induced peroxidation, early apoptosis, and abnormal mitochondrial membrane potential in sperm. Significantly, high-dose OLA impaired in vitro fertilized embryo development, indicated by the decreased percentages of 2-cell and blastocyst formation. Surprisingly, the natural fertility of males was unaffected after OLA gavage, which was indicated by the comparable litter size after mating. However, paternal gavage of OLA significantly decreased the survival rate of the offspring from the age of 4 weeks. In sum, our study showed that OLA gavage in male mice damages sperm quality and offspring survival, illustrating the use of OLA as a feed additive should be strictly restricted.

Effect of spermidine on ameliorating spermatogenic disorders in diabetic mice via regulating glycolysis pathway

Diabetes mellitus (DM), a high incidence metabolic disease, is related to the impairment of male spermatogenic function. Spermidine (SPM), one of the biogenic amines, was identified from human seminal plasma and believed to have multiple pharmacological functions. However, there exists little evidence that reported SPM’s effects on moderating diabetic male spermatogenic function. Thus, the objective of this study was to investigate the SPM’s protective effects on testicular spermatogenic function in streptozotocin (STZ)-induced type 1 diabetic mice. Therefore, 40 mature male C57BL/6 J mice were divided into four main groups: the control group (n = 10), the diabetic group (n = 10), the 2.5 mg/kg SPM-treated diabetic group (n = 10) and the 5 mg/kg SPM-treated diabetic group (n = 10), which was given intraperitoneally for 8 weeks. The type 1 diabetic mice model was established by a single intraperitoneal injection of STZ 120 mg/kg. The results showed that, compare to the control group, the body and testis weight, as well the number of sperm were decreased, while the rate of sperm malformation was significantly increased in STZ-induced diabetic mice. Then the testicular morphology was observed, which showed that seminiferous tubule of testis were arranged in mess, the area and diameter of which was decreased, along with downregulated anti-apoptotic factor (Bcl-2) expression, and upregulated pro-apoptotic factor (Bax) expression in the testes. Furthermore, testicular genetic expression levels of Sertoli cells (SCs) markers (WT1, GATA4 and Vimentin) detected that the pathological changes aggravated observably, such as the severity of tubule degeneration increased. Compared to the saline-treated DM mice, SPM treatment markedly improved testicular function, with an increment in the body and testis weight as well as sperm count. Pro-apoptotic factor (Bax) was down-regulated expression with the up-regulated expression of Bcl-2 and suppression of apoptosis in the testes. What’s more, expression of WT1, GATA4, Vimentin and the expressions of glycolytic rate-limiting enzyme genes (HK2, PKM2, LDHA) in diabetic testes were also upregulated by SPM supplement. The evidence derived from this study indicated that the SMP’s positive effect on moderating spermatogenic disorder in T1DM mice’s testis. This positive effect is delivered via promoting spermatogenic cell proliferation and participating in the glycolytic pathway’s activation.

Effect of obesity on fertility parameters in WIO mice model.

Male infertility is mostly due to low sperm quality, which accounts for about 50% of the causes of infertility. The reasons for low sperm quality are still unclear. Nowadays, many drinks contain high levels of fat, and its effect on fertility is not yet known. To investigate the effect of cholesterol-containing water on male fertility. Forty BALB/c male mice were divided into 2 groups: the control group and the water-induced obesity (WIO) group. Body and testicular weights were recorded and analyzed statistically. Testicular tissues were examined. Serum contents of total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), free testosterone (FT), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were determined. Motility count and morphology of sperm were analyzed. Real-time polymerase chain reaction (RT-PCR) was performed for SYCP3, VEGFA and WT1 genes. The results showed that the WIO group presented the highly significant values for mice body and testis weight, and TC, TG and LDL level in serum (p < 0.05), when compared to the control group. The level of FT, LH and FSH in serum was significantly decreased (p < 0.05) in the WIO group compared with the control group. Seminiferous tubules of testes became thin, and Sertoli cells showed mild atrophy in this group. Also, the count and motility of sperm significantly reduced while the ratio of sperm abnormalities significantly increased in the WIO group compared with the control group (p < 0.05). The results of RT-PCR showed that SYCP3, VEGFA and WT1 genes were significantly downregulated (p < 0.05) in the WIO group compared with the control group. This study indicated that drinks containing high levels of fat may have negative effects on male fertility due to the reduction of the sexual hormones level in serum, the expression of SYCP3, VEGFA and WT1 genes, count and motility of sperm, as well as an increase in sperm abnormalities and pathological changes in the testicular tissues.

Expression and immunolocalisation of PPARγ in reproductive tissues of rabbits fed n-3 PUFA-enriched diets

Background: Since fatty acids are natural ligands of peroxisome proliferator-activated receptors (PPARs), involved in a wide spectrum of metabolic activities, we explored the protein expression and localisation of PPARγ, as well as susceptibility to lipoperoxidation (LPO), in reproductive tissues of rabbits fed n-3 polyunsaturated fatty acid (PUFA)-enriched diets. Methods: Nine rabbit bucks were divided into three groups and fed different diets: FLAX diet supplemented with 10% extruded flaxseed, FISH diet containing 3.5% fish oil and standard diet as control. Protein expression by western blot analysis, localisation of PPARγ by immunofluorescence and susceptibility to LPO by measuring F2-Isoprostanes (F2-IsoPs) were explored. Results: Down-regulation of PPARγ expression was observed in control rabbit testes compared with FLAX (p &lt; 0.01) and FISH (p &lt; 0.05) dietary groups. On the contrary, an up-regulation in rabbit epididymis was observed in both n-3 PUFAs dietary groups compared with the control (p &lt; 0.001). Immunofluorescent staining of PPARγ showed a strong expression in the interstitial tissue and seminiferous tubules of testes from groups fed n-3 PUFA-enriched diets compared to controls; the PPARγ signal was brighter in the epididymis of controls than in those of rabbits fed n-3 PUFA-enriched diets. Interestingly, F2-IsoP levels were significantly higher in testis and epididymal tissue of control compared to n-3 PUFAs dietary groups (p &lt; 0.001). Conclusion: PPARγ is present in male reproductive tissues, in particular in the epididymis and interstitial testicular tissue. PPARγ expression appears to be influenced by n-3 PUFAs dietary sources and to play a role in supporting sperm maturation. n-3 PUFAs diets, reducing F2-IsoP levels, commonly considered proinflammatory molecules linked to oxidative injuries, may also support PPARγ activity.

Pyroglutamylated RFamide peptide (QRFP): Role in early testicular development in relation to Sertoli cell maturation in prepubertal mice

QRFP, an orexigenic neuropeptide, binds to its cognate receptor GPR103 and regulates various biological functions. We have recently shown that QRFP and its receptor are present in mice testes and that their expression is high during early postnatal period. The present study aimed to investigate the effect of sustained high level of QRFP on Sertoli cells proliferation and differentiation and to relate these events with germ cell differentiation and lumen formation in the seminiferous tubules in mice testes during prepubertal period. QRFP was injected intraperitoneally to male mice from postnatal day 5 to 16. Morphometric analysis and various markers related to Sertoli cell maturation (WT1, p27kip1, AMH, AR and CYP19A1) and germ cell proliferation and differentiation (PCNA, GDNF and c-Kit) were evaluated. QRFP administration caused an early lumen formation in the seminiferous tubules in testis of treated mice. Further, there was a significant increase in p27kip1 expression and a marked decrease in AMH expression in QRFP-treated mice compared to controls. However, no appreciable change was noted in AR expression in treated mice. QRFP treatment also caused an increase in c-Kit expression in treated mice compared to controls, suggesting an accelerated spermatogonial differentiation in testis of QRFP-treated mice. Taken together, the present results suggest that the prolonged high level of QRFP increases Sertoli cell maturation, which, in turn, plays a contributory role in increasing the pace of germ cell differentiation and formation of lumen in the seminiferous tubules.

Identification of genomic imbalances (CNVs as well as LOH) in sertoli cell only syndrome cases through cytoscan microarray.

Sertoli cell only syndrome (SCOS) is characterized by complete absence of germ cells in seminiferous tubules of testis. SCOS is multifactorial but genetic factors play a major role in pathogenesis of the disorder with idiopathic origin. Genetic factors majorly include sex chromosomal aneuploidy and Yq Microdeletion. But a large number of cases are still idiopathic. The study aimed to evaluate the genomic imbalances (CNVs and LOH) in idiopathic SCOS patients. The study is based on 28 apparent idiopathic SCOS cases and 10 controls. Molecular cytogenetic techniques viz., FISH, STS-Multiplex PCR and Affymetrix cytoscan microarray (750K) were used. The microarray screened whole genomic imbalances in DNA from peripheral blood of 25 cases (excluded Klinefelter syndrome patients) and testicular FNAC sample of 2 cases. High FSH and low Inhibin B were observed in cases as compared to control controls groups. Four cases of sex chromosomal abnormality (i.e., three non-mosaic 47, XXY males and one non-mosaic 46, XX male) as well as four cases of Yq microdeletion (i.e., three cases with AZFc deletion and one case with complete AZFa, b and c deletion) were identified. Microarray detected unbalanced translocation of two segments of Y-chromosome i.e., Yp11.31-p11.2 (~4.o mb region, involving SRY) and Yp11.2 (~2.5 mb region) on X-chromosome in XX male. Also, loss of segment on same X-chromosome involving PAR1 region was identified. We have identified both autosomal and sex chromosomal CNVs (recurrent as well as private) involving candidate genes like SYCE1, ZFPM2, SRPK1, DAZ1, BPY2, HSFY1, VCY1 etc. All these CNVs are possibly associated with SCOS pathogenesis. CNVs identified in cases were already reported as pathogenic variant in clinical database DECIPHER. Microarray also detected many LOH (all autosomal, >3.0 mb size) that covered genes with spermatogenesis related function. The mechanism of action of LOH in pathogenesis of SCOS still remains unravelled. CNVs and LOH related to spermatogenesis identified from two different sample types (blood vs. testicular tissue) were discordant. This study should be extended for larger cohort of patients.

Dietary supplementation of grape seed tannin extract stimulated testis development, changed fatty acid profiles and increased testis antioxidant capacity in pre-puberty hu lambs

Grape seed tannin extract (GPE) from wine grape pomace has many effective anti-oxidative effects and is used as a promising natural feed additive in the animal feed industry. This study investigated the effect of GPE as a source of tannin on the antioxidant capacity and testis development in Hu lambs. Twenty-seven 3-month-old ram lambs were randomly assigned to three groups. For each treatment group, nine lambs were allocated to nine pens (one lamb per pen). The lambs in the control group were fed a control diet without GPE for 61 days from D21 to D80. Group I (TAN1) was fed with 0.36% GPE diet, and Group II (TAN2) was fed with 0.72% GPE diet. After an 81-day feeding trial, all lambs except the heaviest and lightest in each group were humanely slaughtered and investigated. Results showed that feeding GPE did not affect the body weight, average daily gain, dry matter intake, scrotal circumference, and testis index. Meanwhile, feeding with 0.36% GPE diet increased testis weight, testis volume, and epididymis weight (P ≤ 0.05) compared with those of the control, but no difference was found between TAN1 and TAN2 groups. Copper-zinc superoxide dismutase (Cu-ZnSOD), steroid acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc), follicle-stimulating hormone receptor (FSHR), elongation of very long chain fatty acid protein 2 (ELOVL2), fatty acid desaturase (FADS2), and proliferating cell nuclear antigen (PCNA) mRNA in TAN1 and TAN2 groups were significantly up-regulated (P < 0.05). GPE also markedly increased the antioxidant status of testis. Compared with the control group, the treatment groups showed significantly increased superoxide dismutase (SOD) activity (314.23 ± 18.64 U/mg prot in control, 505.22 ± 63.47 U/mg prot in TAN1 and 587.88 ± 55.94 U/mg prot in TAN2, P < 0.05) and total antioxidant capacity (T-AOC) (98.23 ± 18.99 U/g prot in control, 202.15 ± 34.19 U/g prot in TAN1 and 189.57 ± 18.95 U/g prot in TAN2, P < 0.05). Consuming 0.72% GPE also changed the fatty acid profiles in testis with increased C15:1, C22:6n3, and total n-3 fatty acids (P < 0.05) but decreased C22:5n3 (P < 0.05). Therefore, feeding lambs with GPE stimulated testis seminiferous tubule development and increased the number of Sertoli cells (10.56 ± 0.44 in control, 14.10 ± 0.57 in TAN1 and 13.60 ± 0.42, P < 0.05), and seminiferous tubule diameter (109.30 ± 4.56 μm in control, 164.49 ± 5.37 μm in TAN1 and 146.56 ± 4.53 μm in TAN2, P < 0.05). These results suggested that feeding GPE in the early reproductive development stage of lambs upregulated the expression of antioxidative, steroidogenesis, and polyunsaturated fatty acid metabolism-related genes, changed the fatty acid profiles, increased the antioxidant capacity in lamb's testis, and contributed to testis development and spermatogenesis.

Ethyl acetate fraction of Spathodea campanulata (Bignoniaceae) attenuates lead acetate induced testicular toxicity in male Wistar rats.

The use of Spathodea campanulata in folklore medicine for the management of reproductive disorders has been poorly reported. We sought to investigate the protective potential of the ethyl acetate fraction of S.campanulata stem bark extract (EFSC) on lead acetate-induced (LA) testicular toxicity in male rats. Animals during a 28days treatment received dimethyl sulfoxide (DMSO, 0.1%), LA (20mg/kg), and EFSC (200mg/kg). Others received EFSC only (100, 200, and 400mg/kg) or vitamin E (100mg/kg) 1h prior to LA (20mg/kg) administration. LA administration decreased sperm counts and motility by 36.39 and 40.69% respectively in rats. Also, LA-untreated rats showed elevated malondialdehyde (MDA) and decreased total proteins in testis (260, 33%) and epididymis (62, 29%) respectively. However, EFSC (100, 200, or 400mg/kg) administrations improved sperm morphological characteristics as well as antioxidant status in LA-treated rats. EFSC (400mg/kg) showed improved testis seminiferous tubules that were almost normal in the LA-treated rats. Further, EFSC contains a high 9-octadecenoic acid methyl ester. Overall, evidence by LA-induced testicular toxicity, EFSC provides chemopreventive roles via antioxidant mechanisms.

APPLICATION EFFICIENCY OF BOVINE SERUM ALBUMIN FOR RECOVERY OF SEMINIFEROUS TUBULES OF TESTES AFTER CRYOPRESERVATION

An optimal approach to the recovery of testicular tissue fragments after cryopreservation is critical for their further use in order to successful fertility restoration. Aim. The purpose of this study was to investigate the effect of bovine serum albumin (BSA) addition to the rehabilitation medium on the morphofunctional characteristics of fragments of seminiferous tubules of testes (FSTT) of immature rats after cryopreservation. Methods. The object of the study was cryopreserved by slow cooling and vitrified FSTT. Warmed samples were incubated for 30 min in Leibovitz medium supplemented with BSA at concentrations of 0, 2, 5 or 10%. After that, morphological characteristics, the activity of the metabolic and antioxidant systems were evaluated. Results. It was found that in the samples cryopreserved by slow cooling, the use of 5% BSA contributed to the increase in the safety of spermatogenic epithelium cells, in the levels of metabolic and antioxidant activities. In case of vitrified FSTT samples, it was observed that the addition of 5% BSA to the medium promoted the repair of minor tissue damage and increased metabolic activity level, but did not affect the state of the antioxidant defense system. Conclusions. The obtained data can be used to develop an effective rehabilitation medium for cryopreserved fragments of the seminiferous tubules of testes using BSA.