scholarly journals De novo morphogenesis of testis tissue: an improved bioassay to investigate the role of VEGF165 during testis formation

Reproduction ◽  
2014 ◽  
Vol 148 (1) ◽  
pp. 109-117 ◽  
Author(s):  
Camila Dores ◽  
Ina Dobrinski
Keyword(s):  
Blood Vessel ◽  
Cell Suspension ◽  
Germ Cells ◽  
De Novo ◽  
Donor Cell ◽  
Seminiferous Tubules ◽  
Tissue Formation ◽  
Tubule Formation ◽  
Testis Tissue

De novo formation of testis tissue from single-cell suspensions allows manipulation of different testicular compartments before grafting to study testicular development and the spermatogonial stem cell niche. However, the low percentages of newly formed seminiferous tubules supporting complete spermatogenesis and lack of a defined protocol have limited the use of this bioassay. Low spermatogenic efficiency in de novo formed tissue could result from the scarcity of germ cells in the donor cell suspension, cell damage caused by handling or from hypoxia during tissue formation in the host environment. In this study, we compared different proportions of spermatogonia in the donor cell suspension and the use of Matrigel as a scaffold to support de novo tissue formation and spermatogenesis. Then, we used the system to investigate the role of vascular endothelial growth factor 165 (VEGF165) during testicular morphogenesis on blood vessel and seminiferous tubule formation, and on presence of germ cells in the de novo developed tubules. Our results show that donor cell pellets with 10×106 porcine neonatal testicular cells in Matrigel efficiently formed testis tissue de novo. Contrary to what was expected, the enrichment of the cell suspension with germ cells did not result in higher numbers of tubules supporting spermatogenesis. The addition of VEGF165 did not improve blood vessel or tubule formation, but it enhanced the number of tubules containing spermatogonia. These results indicate that spermatogenic efficiency was improved by the addition of Matrigel, and that VEGF165 may have a protective role supporting germ cell establishment in their niche.

1 XENOGRAFTING OF ADULT MAMMALIAN TESTIS TISSUE

2007 ◽  
Vol 19 (1) ◽  
pp. 119
Author(s):  
L. Arregui ◽  
R. Rathi ◽  
W. Zeng ◽  
A. Honaramooz ◽  
M. Gomendio ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Germ Cell ◽  
Germ Cells ◽  
Sertoli Cells ◽  
Human Testis ◽  
Seminiferous Tubules ◽  
Donor Tissue ◽  
Germ Cell Differentiation ◽  
Sexually Mature ◽  
Testis Tissue

Testis tissue grafting presents an option for preservation of genetic material when sperm recovery is not possible. Grafting of testis tissue from sexually immature males to immunodeficient mice results in germ cell differentiation and production of fertilization-competent sperm from different mammalian species (Honaramooz et al. 2002 Nature 418, 778–781). However, the efficiency of testis tissue xenografting from adult donors has not been critically evaluated. Spermatogenesis was arrested at meiosis in grafts from mature horses (Rathi et al. 2006 Reproduction 131, 1091–1098) and hamsters (Schlatt et al. 2002 Reproduction 124, 339–346), and no germ cell differentiation occurred in xenografts of adult human testis tissue (Schlatt et al. 2006 Hum. Reprod. 21, 384–389). The objective of this study was to investigate survival and germ cell differentiation of testis xenografts from sexually mature donors of different species. Small fragments of testis tissue from 10 donor animals of 5 species were grafted under the back skin of immunodeficient, castrated male mice (n = 37, 2–6/donor). Donors were pig (8 months old), goat (18 months old and 4 years old) (n = 2), bull (3 years old), donkey (13 months old), and rhesus monkey (3, 6, 11, and 12 years old). At the time of grafting, donor tissue contained elongated spermatids, albeit to different degrees (>75% of seminiferous tubules in testis tissue from pig, goat, bull, and 6–12-year-old monkeys, and 33 or 66% of tubules in tissue from donkey or 3-year-old monkey, respectively). Grafts were recovered <12 weeks (n = 14 mice), 12–24 weeks (n = 16 mice), and >24 weeks (n = 7 mice) after grafting and classified histologically as completely degenerated (no tubules found), degenerated tubules (only hyalinized seminiferous tubules observed), or according to the most advanced type of germ cell present. Grafts from pig, goat, bull, and 6–12-year-old monkeys contained >60% degenerated tubules or were completely degenerated at all time points analyzed. In contrast, in grafts from the 3-year-old monkey, only 18% of tubules were degenerated, 14% contained Sertoli cells only, 64% contained meiotic, and 4% haploid germ cells at 24 weeks after grafting. Similarly, donkey testis grafts recovered 12–24 weeks after grafting contained <2% degenerated tubules, 46% of tubules had Sertoli cells only, 45% contained meiotic, and 7% haploid germ cells. These results show that survival and differentiation of germ cells in testis grafts from sexually mature mammalian donors is poor. However, better graft survival and maintenance of spermatogenesis occurred in donor tissue from donkey and 3-year-old monkey that were less mature at the time of grafting. Therefore, species and age-related differences appear to exist with regard to germ cell survival and differentiation in xenografts from adult donors. This work was supported by USDA/CSREES 03-35203-13486, NIH/NCRR 5-R01-RR17359-05, the Spanish Ministry of Education, and Science (BES-2004-4112).

scholarly journals SIRT1 Expression and Regulation in the Primate Testis

2021 ◽  
Vol 22 (6) ◽  
pp. 3207
Author(s):  
Fazal Wahab ◽  
Ignacio Rodriguez Polo ◽  
Rüdiger Behr
Keyword(s):  
Germ Cells ◽  
Rhesus Macaques ◽  
Sirtuin 1 ◽  
Primate Species ◽  
Seminiferous Tubules ◽  
Sirt1 Expression ◽  
Protein Substrates ◽  
Development And Differentiation ◽  
Endocrine Factor

The epigenetic mechanisms controlling germ cell development and differentiation are still not well understood. Sirtuin-1 (SIRT1) is a nicotinamide adenosine dinucleotide (NAD)-dependent histone deacetylase and belongs to the sirtuin family of deacetylases. It catalyzes the removal of acetyl groups from a number of protein substrates. Some studies reported a role of SIRT1 in the central and peripheral regulation of reproduction in various non-primate species. However, testicular SIRT1 expression and its possible role in the testis have not been analyzed in primates. Here, we document expression of SIRT1 in testes of different primates and some non-primate species. SIRT1 is expressed mainly in the cells of seminiferous tubules, particularly in germ cells. The majority of SIRT1-positive germ cells were in the meiotic and postmeiotic phase of differentiation. However, SIRT1 expression was also observed in selected premeiotic germ cells, i.e., spermatogonia. SIRT1 co-localized in spermatogonia with irisin, an endocrine factor specifically expressed in primate spermatogonia. In marmoset testicular explant cultures, SIRT1 transcript levels are upregulated by the addition of irisin as compared to untreated controls explants. Rhesus macaques are seasonal breeders with high testicular activity in winter and low testicular activity in summer. Of note, SIRT1 mRNA and SIRT1 protein expression are changed between nonbreeding (low spermatogenesis) and breeding (high spermatogenesis) season. Our data suggest that SIRT1 is a relevant factor for the regulation of spermatogenesis in primates. Further mechanistic studies are required to better understand the role of SIRT1 during spermatogenesis.

Defective spermatogenesis and testosterone levels in kinase suppressor of Ras1 (KSR1)-deficient mice

2019 ◽  
Vol 31 (8) ◽  
pp. 1369
Author(s):  
Elena Moretti ◽  
Giulia Collodel ◽  
Giuseppe Belmonte ◽  
Daria Noto ◽  
Emanuele Giurisato
Keyword(s):  
Molecular Mechanisms ◽  
Sperm Concentration ◽  
Testicular Tissue ◽  
Seminiferous Tubules ◽  
Interstitial Tissue ◽  
Testosterone Levels ◽  
Transmission Electron ◽  
Human Spermatogenesis ◽  
Testis Tissue

The aim of this study was to clarify the role of the protein kinase suppressor of Ras1 (KSR1) in spermatogenesis. Spermatogenesis in ksr1−/− mice was studied in testicular tissue and epididymal spermatozoa by light and transmission electron microscopy and by immunofluorescence using antibodies to ghrelin and 3β-hydroxysteroid dehydrogenase (3β-HSD). Blood testosterone levels were also assessed. ksr1−/− mice showed reduced epididymal sperm concentration and motility as compared with wild-type (wt) mice. Testis tissue from ksr1−/− mice revealed a prevalent spermatogenetic arrest at the spermatocyte stage; the interstitial tissue was hypertrophic and the cytoplasm of the Leydig cells was full of lipid droplets. Ghrelin signal was present in the seminiferous tubules and, particularly, in the interstitial tissue of wt mice; however, in ksr1−/− mice ghrelin expression was very weak in both the interstitial tissue and tubules. On the contrary, the signal of 3β-HSD was weak in the interstitial tissue of wt and strong in ksr1−/− mice. Testosterone levels were significantly increased in the blood of ksr1−/− mice (P<0.05) as compared with wt. The results obtained reveal the importance of the KSR scaffold proteins in the spermatogenetic process. The study of the molecular mechanisms associated with spermatogenetic defects in a mouse model is essential to understand the factors involved in human spermatogenesis.

scholarly journals The Versatile Role of Matrix Metalloproteinase for the Diverse Results of Fibrosis Treatment

Molecules ◽  
2019 ◽  
Vol 24 (22) ◽  
pp. 4188 ◽  
Author(s):  
Hong-Meng Chuang ◽  
Yu-Shuan Chen ◽  
Horng-Jyh Harn
Keyword(s):  
Extracellular Matrix ◽  
Wound Healing ◽  
Cardiovascular Diseases ◽  
De Novo ◽  
Biological Processes ◽  
Tissue Formation ◽  
Ecm Degradation ◽  
Excessive Secretion ◽  
Wound Tissue

Fibrosis is a type of chronic organ failure, resulting in the excessive secretion of extracellular matrix (ECM). ECM protects wound tissue from infection and additional injury, and is gradually degraded during wound healing. For some unknown reasons, myofibroblasts (the cells that secrete ECM) do not undergo apoptosis; this is associated with the continuous secretion of ECM and reduced ECM degradation even during de novo tissue formation. Thus, matrix metalloproteinases (MMPs) are considered to be a potential target of fibrosis treatment because they are the main groups of ECM-degrading enzymes. However, MMPs participate not only in ECM degradation but also in the development of various biological processes that show the potential to treat diseases such as stroke, cardiovascular diseases, and arthritis. Therefore, treatment involving the targeting of MMPs might impede typical functions. Here, we evaluated the links between these MMP functions and possible detrimental effects of fibrosis treatment, and also considered possible approaches for further applications.

scholarly journals Regeneration of Leydig cells in ectopically autografted adult mouse testes

Reproduction ◽  
2015 ◽  
Vol 149 (3) ◽  
pp. 259-268 ◽  
Author(s):  
Himesh Makala ◽  
Lavanya Pothana ◽  
Surabhi Sonam ◽  
Ashwini Malla ◽  
Sandeep Goel
Keyword(s):  
Germ Cells ◽  
Leydig Cell ◽  
Adult Mouse ◽  
De Novo ◽  
Serum Testosterone ◽  
Serum Testosterone Level ◽  
Seminiferous Tubules ◽  
Testicular Development ◽  
Intrinsic Factors ◽  
Specific Proteins

Ectopic autografting of testis tissue is a promising approach for studying testicular development, male germline preservation and restoration of male fertility. In this study, we examined the fate of various testicular cells in adult mouse testes following ectopic autografting at 1, 2, 4 and 8 weeks post grafting. Histological examination showed no evidence of re-establishment of spermatogenesis in autografts, and progressive degeneration of seminiferous tubules was detected. Expression of germ cell-specific proteins such as POU5F1, DAZL, TNP1, TNP2, PRM1 and PRM2 revealed that, although proliferating and differentiating spermatogenic germ cells such as spermatogonia, spermatocytes and spermatids could survive in autografts until 4 weeks, only terminally differentiated germ cells such as sperm persisted in autografts until 8 weeks. The presence of Sertoli and peritubular myoid cells, as indicated by expression of WT1 and ACTA2 proteins, respectively, was evident in the autografts until 8 weeks. Interestingly, seminal vesicle weight and serum testosterone level were restored in autografted mice by 8 weeks post grafting. The expression of Leydig cell-specific proteins such as CYP11A1, HSD3B2 and LHCGR showed revival of Leydig cell (LC) populations in autografts over time since grafting. Elevated expression of PDGFRA, LIF, DHH and NEFH in autografts indicated de novo regeneration of LC populations. Autografted adult testis can be used as a model for investigating Leydig cell regeneration, steroidogenesis and regulation of the intrinsic factors involved in Leydig cell development. The success of this rodent model can have therapeutic applications for adult human males undergoing sterilizing cancer therapy.

VE-cadherin is not required for the formation of nascent blood vessels but acts to prevent their disassembly

Blood ◽  
2005 ◽  
Vol 105 (7) ◽  
pp. 2771-2776 ◽  
Author(s):  
Christopher V. Crosby ◽  
Paul A. Fleming ◽  
W. Scott Argraves ◽  
Monica Corada ◽  
Lucia Zanetta ◽  
...  
Keyword(s):  
Blood Vessel ◽  
Blood Vessels ◽  
Cell Adhesion Molecule ◽  
De Novo ◽  
Temporal Correlation ◽  
Vascular Endothelial ◽  
Cell Clusters ◽  
Platelet Endothelial Cell Adhesion ◽  
Specific Step

Abstract We investigated the role of vascular endothelial (VE)–cadherin in blood vessel morphogenesis and established a temporal correlation linking the failure in vessel morphogenesis in VE-cadherin null embryos to a specific step in vasculogenesis. We showed that the sequence in which blood vessels failed followed the order in which they had formed (ie, those forming first—yolk sac, allantoic and endocardial vessels—were the first to display morphologic abnormalities). We next showed that in place of normal reticulated networks of blood vessels, clusters of platelet endothelial cell adhesion molecule–positive (PECAM+) cells formed within cultured allantois explants from VE-cadherin null embryos. Similarly, a function-blocking VE-cadherin antibody, BV13, caused PECAM+ cell clusters to form in cultured allantois explants from normal mice. Finally, we demonstrated that formation of PECAM+ cell clusters in response to BV13 was not due to a disruption in the formation of nascent vessels but was due to the actual disassembly of nascent vessels. Based on these findings, we conclude that the events of de novo blood vessel formation up to the point at which a vascular epithelium forms (ie, nascent vessels with lumens) are not dependent on VE-cadherin and that VE-cadherin, whose expression is up-regulated following vascular epithelialization, is required to prevent the disassembly of nascent blood vessels.

scholarly journals Maintenance DNA methylation in pre-meiotic germ cells regulates meiotic prophase by facilitating homologous chromosome pairing

Development ◽  
2021 ◽  
Vol 148 (10) ◽  
Author(s):  
Yuki Takada ◽  
Ruken Yaman-Deveci ◽  
Takayuki Shirakawa ◽  
Jafar Sharif ◽  
Shin-ichi Tomizawa ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Germ Cells ◽  
Meiotic Prophase ◽  
De Novo ◽  
Pericentric Heterochromatin ◽  
Male Germ Cells ◽  
Homologous Chromosomes ◽  
Homologous Chromosome Pairing ◽  
Synaptic Failure

ABSTRACT Heterochromatin-related epigenetic mechanisms, such as DNA methylation, facilitate pairing of homologous chromosomes during the meiotic prophase of mammalian spermatogenesis. In pro-spermatogonia, de novo DNA methylation plays a key role in completing meiotic prophase and initiating meiotic division. However, the role of maintenance DNA methylation in the regulation of meiosis, especially in the adult, is not well understood. Here, we reveal that NP95 (also known as UHRF1) and DNMT1 – two essential proteins for maintenance DNA methylation – are co-expressed in spermatogonia and are necessary for meiosis in male germ cells. We find that Np95- or Dnmt1-deficient spermatocytes exhibit spermatogenic defects characterized by synaptic failure during meiotic prophase. In addition, assembly of pericentric heterochromatin clusters in early meiotic prophase, a phenomenon that is required for subsequent pairing of homologous chromosomes, is disrupted in both mutants. Based on these observations, we propose that DNA methylation, established in pre-meiotic spermatogonia, regulates synapsis of homologous chromosomes and, in turn, quality control of male germ cells. Maintenance DNA methylation, therefore, plays a role in ensuring faithful transmission of both genetic and epigenetic information to offspring.

scholarly journals MORC3, a novel MIWI2 association partner, as an epigenetic regulator of piRNA dependent transposon silencing in male germ cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kanako Kojima-Kita ◽  
Satomi Kuramochi-Miyagawa ◽  
Manabu Nakayama ◽  
Haruhiko Miyata ◽  
Steven E. Jacobsen ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Germ Cells ◽  
De Novo ◽  
Effector Proteins ◽  
P Element ◽  
Male Germ Cells ◽  
Transposon Silencing ◽  
Association Partner ◽  
Pirna Pathway

AbstractThe PIWI (P-element-induced wimpy testis)-interacting-RNA (piRNA) pathway plays a crucial role in the repression of TE (transposable element) expression via de novo DNA methylation in mouse embryonic male germ cells. Various proteins, including MIWI2 are involved in the process. TE silencing is ensured by piRNA-guided MIWI2 that recruits some effector proteins of the DNA methylation machinery to TE regions. However, the molecular mechanism underlying the methylation is complex and has not been fully elucidated. Here, we identified MORC3 as a novel associating partner of MIWI2 and also a nuclear effector of retrotransposon silencing via piRNA-dependent de novo DNA methylation in embryonic testis. Moreover, we show that MORC3 is important for transcription of piRNA precursors and subsequently affects piRNA production. Thus, we provide the first mechanistic insights into the role of this effector protein in the first stage of piRNA biogenesis in embryonic TE silencing mechanism.

309 THE SPERMATOGENIC CYCLE IN MAMMALIAN TESTIS XENOGRAFTS

2006 ◽  
Vol 18 (2) ◽  
pp. 262
Author(s):  
W. Zeng ◽  
G. F. Avelar ◽  
R. Rathi ◽  
L. R. Franca ◽  
I. Dobrinski
Keyword(s):  
Germ Cells ◽  
Stage I ◽  
Stage Ii ◽  
Brdu Incorporation ◽  
Seminiferous Tubules ◽  
Sperm Production ◽  
Porcine Tissue ◽  
Brdu Injection ◽  
Donor Species ◽  
Testis Tissue

Grafting of immature testis tissue from different mammalian donor species into mouse hosts results in production of spermatozoa from the donor species. Xenografting of testis tissue from rhesus monkeys, pigs, and sheep accelerates sperm production. To determine whether this shortened time to sperm production is due to the reduced spermatogenic cycle length, we applied bromodeoxyuridine (BrdU) incorporation to analyze the spermatogenic cycle in porcine and ovine testis xenografts. Testes from 1-2-week-old Yorkshire cross pigs and 1-week-old Suffolk sheep were cut into small fragments (approximately 1 � 1 � 2 mm) and eight fragments were grafted under the back skin of each castrated male immunodeficient NCR nude recipient mouse (n = 7 for pig, n = 5 for sheep). Mice were given BrdU (100 mg/kg i.p.) at 7 months (porcine tissue) or 6 months (ovine tissue) post-transplantation. Mice carrying porcine tissue were sacrificed 1 h, 9 days or 18 days after BrdU injection. Mice with ovine testicular tissue were sacrificed 1 h, 11 days or 22 days after BrdU injection. Analysis time points were chosen based on the reported length of the spermatogenic cycle in pigs and sheep (approximately 9 days and 11 days, respectively). All eight stages of the spermatogenic cycle were analyzed to identify the most advanced germ cells labeled in each time period after BrdU injection. All seminiferous tubules containing full spermatogenesis were analyzed. Histologically, 51.8% (range 7 to 98%, n = 2040 tubules) of seminiferous tubules from porcine grafts, and 64.4% (range 2 to 92%, n = 2903 tubules) of seminiferous tubules from ovine grafts presented complete spermatogenesis. In porcine grafts, the most advanced germ cells labeled 1 h after BrdU injection were primary spermatocytes in pre-leptotene/leptotene at stage I of the spermatogenic cycle. At 9 days and 18 days after injection, the most advanced labeled germ cells were primary spermatocytes at pachytene at stage I and elongating spermatids at late stage II, respectively. In ovine grafts, the most advanced labeled germ cells at 1 h, 11 days and 22 days were pre-leptotene/leptotene at stage II, primary spermatocytes at the pachytene at stage I and elongating spermatids at stage II, respectively. These results indicate that each spermatogenic cycle in porcine and ovine testis xenografts lasts around 9 days and 11 days, respectively. Therefore, the length of the spermatogenic cycle is conserved in porcine and ovine testis xenografts and shortened time to sperm production is likely due to accelerated maturation of the testicular somatic components, such as Sertoli cells. This work was supported by NIH R01 RR17359-01.

scholarly journals The insulin sensitiser metformin regulates chicken Sertoli and germ cell populations

Reproduction ◽  
2016 ◽  
Vol 151 (5) ◽  
pp. 527-538 ◽  
Author(s):  
M Faure ◽  
E Guibert ◽  
S Alves ◽  
B Pain ◽  
C Ramé ◽  
...  
Keyword(s):  
Germ Cells ◽  
Sertoli Cells ◽  
Energy Content ◽  
Morphological Changes ◽  
Seminiferous Tubules ◽  
Reduce Food Intake ◽  
Testicular Weight

Abstract Metformin, an insulin sensitiser from the biguanide family of molecules, is used for the treatment of insulin resistance in type 2 diabetes individuals. It increases peripheral glucose uptake and may reduce food intake. Based on the tight link between metabolism and fertility, we investigated the role of metformin on testicular function using in vitro culture of Sertoli cells and seminiferous tubules, complemented by in vivo data obtained following metformin administration to prepubertal chickens. In vitro, metformin treatment reduced Sertoli cell proliferation without inducing apoptosis and morphological changes. The metabolism of Sertoli cells was affected because lactate secretion by Sertoli cells increased approximately twofold and intracellular free ATP was negatively impacted. Two important pathways regulating proliferation and metabolism in Sertoli cells were assayed. Metformin exposure was not associated with an increased phosphorylation of AKT or ERK. There was a 90% reduction in the proportion of proliferating germ cells after a 96-h exposure of seminiferous tubule cultures to metformin. In vivo, 6-week-old chickens treated with metformin for 3 weeks exhibited reduced testicular weight and a 50% decrease in testosterone levels. The expression of a marker of undifferentiated germ cells was unchanged in contrast to the decrease in expression of ‘protamine’, a marker of differentiated germ cells. In conclusion, these results suggest that metformin affects the testicular energy content and the proliferative ability of Sertoli and germ cells. Reproduction (2016) 151 527–538

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