scholarly journals Multi Species Analyses Reveal Testicular T3 Metabolism and Signalling as a Target of Environmental Pesticides

Cells ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2187
Author(s):  
Valeria Nittoli ◽  
Marco Colella ◽  
Alfonsina Porciello ◽  
Carla Reale ◽  
Luca Roberto ◽  
...  
Keyword(s):  
Male Fertility ◽  
Ex Vivo ◽  
Optimal Level ◽  
Seminiferous Tubules ◽  
Biological Processes ◽  
Developmental Exposure ◽  
Germ Cell Differentiation ◽  
Species Analysis ◽  
Ethylene Thiourea ◽  
Reproductive Capability

Thyroid hormones (THs) regulate many biological processes in vertebrates, including reproduction. Testicular somatic and germ cells are equipped with the arrays of enzymes (deiodinases), transporters, and receptors necessary to locally maintain the optimal level of THs and their signalling, needed for their functions and spermatogenesis. Pesticides, as chlorpyrifos (CPF) and ethylene thiourea (ETU), impair the function of thyroid and testis, affecting male fertility. However, their ability to disarrange testicular T3 (t-T3) metabolism and signalling is poorly considered. Here, a multi-species analysis involving zebrafish and mouse suggests the damage of t-T3 metabolism and signalling as a mechanism of gonadic toxicity of low-doses CPF and ETU. Indeed, the developmental exposure to both compounds reduces Dio2 transcript in both models, as well as in ex-vivo cultures of murine seminiferous tubules, and it is linked to alteration of steroidogenesis and germ cell differentiation. A major impact on spermatogonia was confirmed molecularly by the expression of their markers and morphologically evidenced in zebrafish. The results reveal that in the adopted models, exposure to both pesticides alters the t-T3 metabolism and signalling, affecting the reproductive capability. Our data, together with previous reports suggest zebrafish as an evaluable model in assessing the action of compounds impairing locally T3 signalling.

scholarly journals Influence of long-term dietary administration of procymidone, a fungicide with anti-androgenic effects, or the phytoestrogen genistein to rats on the pituitary–gonadal axis and Leydig cell steroidogenesis

2005 ◽  
Vol 187 (1) ◽  
pp. 117-124 ◽  
Author(s):  
K Svechnikov ◽  
V Supornsilchai ◽  
M-L Strand ◽  
A Wahlgren ◽  
D Seidlova-Wuttke ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Serum Level ◽  
Leydig Cell ◽  
Leydig Cells ◽  
Ex Vivo ◽  
Elevated Serum ◽  
Seminiferous Tubules ◽  
Dibutyryl Camp

Procymidone is a fungicide with anti-androgenic properties, widely used to protect fruits from fungal infection. Thereby it contaminates fruit products prepared for human consumption. Genistein-containing soy products are increasingly used as food additives with health-promoting properties. Therefore we examined the effects of long-term dietary administration (3 months) of the anti-androgen procymidone (26.4 mg/animal per day) or the phytoestrogen genistein (21.1 mg/animal per day) to rats on the pituitary-gonadal axis in vivo, as well as on Leydig cell steroidogenesis and on spermatogenesis ex vivo. The procymidone-containing diet elevated serum levels of LH and testosterone and, furthermore, Leydig cells isolated from procymidone-treated animals displayed an enhanced capacity for producing testosterone in response to stimulation by hCG or dibutyryl cAMP, as well as elevated expression of steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage (P450 scc) and cytochrome P450 17α (P450c17). In contrast, the rate of DNA synthesis during stages VIII and IX of spermatogenesis in segments of seminiferous tubules isolated from genistein-treated rats was decreased without accompanying changes in the serum level of either LH or testosterone. Nonetheless, genistein did suppress the ex vivo steroidogenic response of Leydig cells to hCG or dibutyryl cAMP by down-regulating their expression of P450 scc. Considered together, our present findings demonstrate that long-term dietary administration of procymidone or genistein to rats exerts different effects on the pituitary–gonadal axis in vivo and on Leydig cell steroidogenesis ex vivo. Possibly as a result of disruption of hormonal feedback control due to its anti-androgenic action, procymidone activates this endocrine axis, thereby causing hyper-gonadotropic activation of testicular steroidogenesis. In contrast, genistein influences spermatogenesis and significantly inhibits Leydig cell steroidogenesis ex vivo without altering the serum level of either LH or testosterone.

169. THE MECHANISM OF SPERMATID MATURATION - A LINK TO TUMOUR SUPPRESSION

2010 ◽  
Vol 22 (9) ◽  
pp. 87
Author(s):  
D. Jamsai ◽  
S. J. Smith ◽  
A. E. O'Connor ◽  
D. J. Merriner ◽  
C. Borg ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Male Fertility ◽  
Rna Binding ◽  
Ex Vivo ◽  
Round Spermatid ◽  
Null Alleles ◽  
Compound Heterozygous ◽  
Binding Motif ◽  
Rna Recognition Motif

To comprehensively uncover novel male fertility regulators, we utilised an unbiased forward genetic screen, ENU mutagenesis. Using this approach, we have identified several novel infertile mouse lines including a male-specific infertile line that we designated ‘Joey’. The mutant Joey mice produced no sperm due to an arrest of male germ cells at the round spermatid stage. The mutation was identified in the RNA binding motif 5 (Rbm5) gene that resulted in an arginine to proline substitution within a highly conserved RNA recognition motif of the protein. The substitution of proline is likely to interfere with RNA binding and/or recognition. In humans, the RBM5 gene maps to a region that is frequently deleted in lung cancers. Ex vivo studies have suggested that RBM5 is a tumour suppressor, apoptosis modulator and RNA splicing regulator. To date, the role of Rbm5 has never been liked to male fertility and the Joey line is the only mouse model of Rbm5 dysfunction. Using our RBM5-specific antibody, we showed that RBM5 is expressed in pachytene spermatocytes and round spermatids. Based on the protein localisation, the proposed role of RBM5 in mRNA processing, the onset of the Joey phenotype, and the site of the identified mutation, we hypothesise that the Rbm5 mutant allele results in a hypomorphic protein, and that RBM5 has an essential role in regulating male germ cell mRNA storage, transport and/or translational regulation of mRNAs that are critical for spermatid maturation. Further, we generated mice compound heterozygous of the Joey Rbm5 mutation and Rbm5 null alleles. We showed that the compound heterozygous males are infertile due to spermatid maturation arrest resembling the Joey mutant males. This result further confirmed the identification of the Rbm5 mutation as a cause of infertility in the Joey mice and a crucial role of Rbm5 in male fertility.

scholarly journals Exosome: A New Player in Translational Nanomedicine

2020 ◽  
Vol 9 (8) ◽  
pp. 2380 ◽  
Author(s):  
Houssam Aheget ◽  
María Tristán-Manzano ◽  
Loubna Mazini ◽  
Marina Cortijo-Gutierrez ◽  
Pablo Galindo-Moreno ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Cell Types ◽  
Structural Features ◽  
Biologically Active ◽  
Biological Processes ◽  
New Approach ◽  
Current State ◽  
Exosome Biogenesis ◽  
Production Techniques

Summary: Exosomes are extracellular vesicles released by the vast majority of cell types both in vivo and ex vivo, upon the fusion of multivesicular bodies (MVBs) with the cellular plasma membrane. Two main functions have been attributed to exosomes: their capacity to transport proteins, lipids and nucleic acids between cells and organs, as well as their potential to act as natural intercellular communicators in normal biological processes and in pathologies. From a clinical perspective, the majority of applications use exosomes as biomarkers of disease. A new approach uses exosomes as biologically active carriers to provide a platform for the enhanced delivery of cargo in vivo. One of the major limitations in developing exosome-based therapies is the difficulty of producing sufficient amounts of safe and efficient exosomes. The identification of potential proteins involved in exosome biogenesis is expected to directly cause a deliberate increase in exosome production. In this review, we summarize the current state of knowledge regarding exosomes, with particular emphasis on their structural features, biosynthesis pathways, production techniques and potential clinical applications.

Human Platelet-Derived Factors Regulate Mesenchymal Stem Cell Gene Expression.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4255-4255
Author(s):  
Christina Malischnik ◽  
Katharina Schallmoser ◽  
Eva Rohde ◽  
Andreas Reinisch ◽  
Christian Guelly ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Growth Factors ◽  
Human Platelet ◽  
Ex Vivo ◽  
Expression Profiles ◽  
Cell Structure ◽  
Considerable Proportion ◽  
Biological Processes ◽  
Regulated Gene Expression

Abstract The use of animal-derived products during human stem cell processing bears the evident risk of xenogeneic prion, virus, or zoonose contamination. Human platelet lysate (HPL) has recently been recognized as a rich source of cytokines and growth factors with the potential to replace fetal bovine serum (FBS) during ex vivo stem cell manipulation. In this study we compared the gene expression profile of human multipotent mesenchymal stromal/stem cells (MSC) during ex vivo expansion for clinical applications under the aegis of either FBS or HPL. The Applied Biosystems 1700 Expression Array System was used for full genome expression profiling of MSC after a 12–14 day expansion period in a previously optimized low density expansion system. Data have been obtained from biological as well as technical replicates. A starting amount of 40μg total RNA was directly labeled and DIG-labeled cDNA was hybridized to Human Genome Survey Microarray V2.0. Attribution of regulated genes to biological processes and pathways was done using the PANTHER® db analysis software. We identified more than 300 genes that are differentially regulated upon culture of MSC in HPL compared to FBS. Biological processes specifically activated in HPL culture include mesoderm development, cell cycle control, hematopoiesis and angiogenesis which interestingly correspond to a considerable proportion of the regenerative function of MSC. In contrast, processes related to cell adhesion and adhesion-mediated signaling, cell structure, cell motility and cell communication are significantly upregulated in MSC after FBS in comparison to HPL culture. Replacing FBS with HPL not only avoids bovine prion, viral and zoonose contamination of MSC for clinical use. The tightly regulated gene expression profiles under the aegis of human growth factors and cytokines provided by HPL may even help to develop new stem cell therapy strategies.

scholarly journals Rhox13 is required for a quantitatively normal first wave of spermatogenesis in mice

Reproduction ◽  
2016 ◽  
Vol 152 (5) ◽  
pp. 379-388 ◽  
Author(s):  
Jonathan T Busada ◽  
Ellen K Velte ◽  
Nicholas Serra ◽  
Kenneth Cook ◽  
Bryan A Niedenberger ◽  
...  
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Histological Analysis ◽  
Gene Knockout ◽  
Cell Types ◽  
Seminiferous Tubules ◽  
Germ Cell Differentiation ◽  
Sperm Counts ◽  
Full Complement ◽  
Specific Association

We previously described a novel germ cell-specific X-linkedreproductivehomeoboxgene (Rhox13) that is upregulated at the level of translation in response to retinoic acid (RA) in differentiating spermatogonia and preleptotene spermatocytes. We hypothesize that RHOX13 plays an essential role in male germ cell differentiation, and have tested this by creating aRhox13gene knockout (KO) mouse.Rhox13KO mice are born in expected Mendelian ratios, and adults have slightly reduced testis weights, yet a full complement of spermatogenic cell types. Young KO mice (at ~7–8 weeks of age) have a ≈50% reduction in epididymal sperm counts, but numbers increased to WT levels as the mice reach ~17 weeks of age. Histological analysis of testes from juvenile KO mice reveals a number of defects during the first wave of spermatogenesis. These include increased apoptosis, delayed appearance of round spermatids and disruption of the precise stage-specific association of germ cells within the seminiferous tubules. Breeding studies reveal that both young and aged KO males produce normal-sized litters. Taken together, our results indicate that RHOX13 is not essential for mouse fertility in a controlled laboratory setting, but that it is required for optimal development of differentiating germ cells and progression of the first wave of spermatogenesis.

scholarly journals Novel Expression and Direct Effects of Adiponectin in the Rat Testis

Endocrinology ◽  
2008 ◽  
Vol 149 (7) ◽  
pp. 3390-3402 ◽  
Author(s):  
J. E. Caminos ◽  
R. Nogueiras ◽  
F. Gaytán ◽  
R. Pineda ◽  
C. R. González ◽  
...  
Keyword(s):  
Hormonal Regulation ◽  
Ex Vivo ◽  
Seminiferous Tubules ◽  
Peroxisome Proliferator ◽  
Direct Effects ◽  
Peroxisome Proliferator Activated Receptor ◽  
Rat Testis ◽  
Protein Levels ◽  
Endocrine Organs

Adiponectin is an adipocyte hormone, with relevant roles in lipid metabolism and glucose homeostasis, recently involved in the control of different endocrine organs, such as the placenta, pituitary and, likely, the ovary. However, whether as described previously for other adipokines, such as leptin and resistin, adiponectin is expressed and/or conducts biological actions in the male gonad remains unexplored. In this study, we provide compelling evidence for the expression, putative hormonal regulation, and direct effects of adiponectin in the rat testis. Testicular expression of adiponectin was demonstrated along postnatal development, with a distinctive pattern of RNA transcripts and discernible protein levels that appeared mostly located at interstitial Leydig cells. Testicular levels of adiponectin mRNA were marginally regulated by pituitary gonadotropins but overtly modulated by metabolic signals, such as glucocorticoids, thyroxine, and peroxisome proliferator-activated receptor-γ, whose effects were partially different from those on circulating levels of adiponectin. In addition, expression of the genes encoding adiponectin receptor (AdipoR)-1 and AdipoR2 was detected in the rat testis, with developmental changes and gonadotropin regulation for AdipoR2 mRNA, and prominent levels of AdipoR1 in seminiferous tubules. Moreover, recombinant adiponectin significantly inhibited basal and human choriogonadotropin-stimulated testosterone secretion ex vivo, whereas it failed to change relative levels of several Sertoli cell-expressed mRNAs, such as stem cell factor and anti-Müllerian hormone. In summary, our data are the first to document the expression, regulation and functional role of adiponectin in the rat testis. Taken together with its recently reported expression in the ovary and its effects on LH secretion and ovarian steroidogenesis, these results further substantiate a multifaceted role of adiponectin in the control of the reproductive axis, which might operate as endocrine integrator linking metabolism and gonadal function.

scholarly journals SPATA33 localizes calcineurin to the mitochondria and regulates sperm motility in mice

2021 ◽  
Vol 118 (35) ◽  
pp. e2106673118
Author(s):  
Haruhiko Miyata ◽  
Seiya Oura ◽  
Akane Morohoshi ◽  
Keisuke Shimada ◽  
Daisuke Mashiko ◽  
...  
Keyword(s):  
Immune Responses ◽  
Sperm Motility ◽  
Knockout Mice ◽  
In Silico ◽  
Male Fertility ◽  
Mitochondrial Protein ◽  
Biological Processes ◽  
Consensus Motif ◽  
Calcium Dependent ◽  
Specific Inhibitors

Calcineurin is a calcium-dependent phosphatase that plays roles in a variety of biological processes including immune responses. In spermatozoa, there is a testis-enriched calcineurin composed of PPP3CC and PPP3R2 (sperm calcineurin) that is essential for sperm motility and male fertility. Because sperm calcineurin has been proposed as a target for reversible male contraceptives, identifying proteins that interact with sperm calcineurin widens the choice for developing specific inhibitors. Here, by screening the calcineurin-interacting PxIxIT consensus motif in silico and analyzing the function of candidate proteins through the generation of gene-modified mice, we discovered that SPATA33 interacts with sperm calcineurin via a PQIIIT sequence. Spata33 knockout mice exhibit reduced sperm motility because of an inflexible midpiece, leading to impaired male fertility, which phenocopies Ppp3cc and Ppp3r2 knockout mice. Further analysis reveals that sperm calcineurin disappears from the mitochondria in the Spata33 knockout testis. In addition, immunoprecipitation analysis indicates that sperm calcineurin interacts with not only SPATA33 but also the mitochondrial protein VDAC2. These results indicate that SPATA33 localizes calcineurin to the mitochondria and regulates sperm motility.

scholarly journals A Protein Engineering Approach for Uncovering Cryptic Ubiquitin-binding Sites: from a Ubiquitin-Variant Inhibitor of APC/C to K48 Chain Binding

2019 ◽  
Author(s):  
Edmond R. Watson ◽  
Christy R. R. Grace ◽  
Wei Zhang ◽  
Darcie J. Miller ◽  
Iain F. Davidson ◽  
...  
Keyword(s):  
Binding Sites ◽  
Protein Quality ◽  
Ex Vivo ◽  
Regulatory Proteins ◽  
Biological Processes ◽  
Anaphase Promoting Complex ◽  
C Terminus ◽  
Ubiquitin Binding ◽  
Enzymatic Cascades

ABSTRACTUbiquitin-mediated proteolysis is a fundamental mechanism used by eukaryotic cells to maintain homeostasis and protein quality, and to control timing in biological processes. Two essential aspects of ubiquitin regulation are conjugation through E1-E2-E3 enzymatic cascades, and recognition by ubiquitin-binding domains. An emerging theme in the ubiquitin field is that these two properties are often amalgamated in conjugation enzymes. In addition to covalent thioester linkage to ubiquitin’s C-terminus for ubiquitin transfer reactions, conjugation enzymes often bind non-covalently and weakly to ubiquitin at “exosites”. However, identification of such sites is typically empirical and particularly challenging in large molecular machines. Here, studying the 1.2 MDa E3 ligase Anaphase-Promoting Complex/Cyclosome (APC/C), which controls cell division and many aspects of neurobiology, we discover a method for identifying unexpected ubiquitin-binding sites. Using a panel of ubiquitin variants (UbVs) we identify a protein-based inhibitor that blocks ubiquitin ligation to APC/C substrates in vitro and ex vivo. Biochemistry, NMR, and cryo EM structurally define the UbV interaction, explain its inhibitory activity through binding the surface on the APC2 subunit that recruits the E2 enzyme UBE2C, and ultimately reveal that this APC2 surface is also a ubiquitin-binding exosite with preference for K48-linked chains. The results provide a new tool for probing APC/C activity, have implications for the coordination of K48-linked Ub chain binding by APC/C with the multistep process of substrate polyubiquitylation, and demonstrate the power of UbV technology for identifying cryptic ubiquitin binding sites within large multiprotein complexes.SIGNIFICANCE STATEMENTUbiquitin-mediated interactions influence numerous biological processes. These are often transient or a part of multivalent interactions. Therefore, unmasking these interactions remains a significant challenge for large, complicated enzymes such as the Anaphase-Promoting Complex/Cyclosome (APC/C), a multisubunit RING E3 ubiquitin (Ub) ligase. APC/C activity regulates numerous facets of biology by targeting key regulatory proteins for Ub-mediated degradation. Using a series of Ub variants (UbVs), we identified a new Ub-binding site on the APC/C that preferentially binds to K48-linked Ub chains. More broadly, we demonstrate a workflow that can be exploited to uncover Ub-binding sites within ubiquitylation machinery and other associated regulatory proteins to interrogate the complexity of the Ub code in biology.

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

2021 ◽  
Vol 22 (20) ◽  
pp. 11157
Author(s):  
Yulia Michailov ◽  
Ali AbuMadighem ◽  
Eitan Lunenfeld ◽  
Joseph Kapelushnik ◽  
Mahmoud Huleihel
Keyword(s):  
Male Fertility ◽  
Inflammatory Cytokine ◽  
Sperm Parameters ◽  
Seminiferous Tubules ◽  
Testis Weight ◽  
Post Injection ◽  
Colony Stimulating Factor ◽  
Expression Levels ◽  
First Time ◽  
Stimulating Factor

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.

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