193 TESTIS TISSUE XENOGRAFTING TO PRESERVE GERM CELLS FROM A CLONED BANTENG CALF

In April 2003, two banteng (Bos javonicus) calves were born after heterologous nuclear transfer of donor cells from a genetically valuable individual frozen in 1978. One of the cloned banteng calves died at one week of age. The calf was found to have one scrotal and one abdominally cryptorchid testis. In an attempt to preserve male germ cells from this valuable animal, parts of each testis were shipped on ice to the University of Pennsylvania for xenografting. Grafting of testis tissue from immature domestic animals and monkeys under the back skin of immunodeficient mice can result in complete spermatogenesis, albeit with different levels of efficiency in different species. The objective of this experiment was to investigate if grafting of immature banteng testis tissue would result in spermatogenesis in a mouse host. Small fragments of tissue (about 1 mm, 3 each) from both testes were grafted under the back skin (4 pieces of scrotal testis on the right side and 4 pieces of retained testis on the left side) of 6 castrated male immunodeficient mice. Histological examination of the testis xenografts was performed 3, 6, 9, 12, and 15 months after transplantation. Weight of the seminal vesicles in the host mouse was recorded as an indicator of bioactive testosterone produced by the xenografts. At the time of grafting, both testes contained seminiferous cords with immature Sertoli cells and gonocytes. At 3, 6, and 9 months after grafting, pachytene spermatocytes were present in the xenografts of the scrotal testis whereas no germ cell differentiation was observed in grafts from the retained testis. However, spermatogenesis in grafts of the scrotal testis did not proceed further through meiosis in grafts analyzed at 12 and 15 months after grafting, with pachytene spermatocytes still the most advanced germ cell type present in grafts recovered 15 months after grafting. The weight of the seminal vesicles in the castrated host mice was restored to pre-castration values showing that xenografts were releasing bioactive testosterone. These results indicate that banteng spermatogenesis was initiated in the mouse host but became arrested at meiosis as observed previously in xenografts of immature bovine or equine testis. Therefore, haploid germ cells could not be recovered. This represents the first example of trying to preserve fertility from a rare, valuable newborn animal by testis tissue xenografting. While xenografting presents a previously unavailable option for preservation of male germ cells from immature individuals, the efficiency of sperm production in testis xenografts appears to be variable and has to be determined empirically for different donor species. This work was supported by USDA 03-35203-13486.

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Germ cell development in equine testis tissue xenografted into mice

Reproduction ◽

10.1530/rep.1.01101 ◽

2006 ◽

Vol 131 (6) ◽

pp. 1091-1098 ◽

Cited By ~ 75

Author(s):

R Rathi ◽

A Honaramooz ◽

W Zeng ◽

R Turner ◽

I Dobrinski

Keyword(s):

Germ Cell ◽

Germ Cells ◽

Meiotic Division ◽

Seminal Vesicles ◽

Post Transplantation ◽

Germ Cell Development ◽

Immunodeficient Mice ◽

Low Efficiency ◽

Underlying Mechanisms ◽

Testis Tissue

Grafting of testis tissue from immature animals to immunodeficient mice results in complete spermatogenesis, albeit with varying efficiency in different species. The objectives of this study were to investigate if grafting of horse testis tissue would result in spermatogenesis, and to assess the effect of exogenous gonadotropins on xenograft development. Small fragments of testis tissue from 7 colts (2 week to 4 years of age) were grafted under the back skin of castrated male immunodeficient mice. For 2 donor animals, half of the mice were treated with gonadotropins. Xenografts were analyzed at 4 and 8 months post-transplantation. Spermatogenic differentiation following grafting ranged from no differentiation to progression through meiosis with appearance of haploid cells. Administration of exogenous gonadotropins appeared to support post-meiotic differentiation. For more mature donor testis samples where spermatogenesis had progressed into or through meiosis, after grafting an initial loss of differentiated germ cells was observed followed by a resurgence of spermatogenesis. However, if haploid cells had been present prior to grafting, spermatogenesis did not progress beyond meiotic division. In all host mice with spermatogenic differentiation in grafts, increased weight of the seminal vesicles compared to castrated mice showed that xenografts were releasing testosterone. These results indicate that horse spermatogenesis occurs in a mouse host albeit with low efficiency. In most cases, spermatogenesis arrested at meiosis. The underlying mechanisms of this spermatogenic arrest require further investigation.

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Gonadal status of male recipient mice influences germ cell development in immature buffalo testis tissue xenograft

Reproduction ◽

10.1530/rep-11-0286 ◽

2012 ◽

Vol 143 (1) ◽

pp. 59-69 ◽

Cited By ~ 12

Author(s):

Niranjan Reddy ◽

Ranjeet Singh Mahla ◽

Revanth Thathi ◽

Sanjay Kumar Suman ◽

Jedy Jose ◽

...

Keyword(s):

Cell Differentiation ◽

Germ Cell ◽

Germ Cells ◽

Serum Testosterone ◽

Intact Mouse ◽

Germ Cell Differentiation ◽

Pachytene Spermatocytes ◽

Gonadal Status ◽

Tubule Diameter ◽

Testis Tissue

Growth and development of immature testis xenograft from various domestic mammals has been shown in mouse recipients; however, buffalo testis xenografts have not been reported to date. In this study, small fragments of testis tissue from 8-week-old buffalo calves were implanted subcutaneously onto the back of immunodeficient male mouse recipients, which were either castrated or left intact (non-castrated). The xenografts were retrieved and analyzed 12 and 24 weeks later. The grafted tissue survived and grew in both types of recipient with a significant increase in weight and seminiferous tubule diameter. Recovery of grafts from intact recipients 24 weeks post-grafting was significantly lower than that from the castrated recipients. Seminal vesicle indices and serum testosterone levels were lower in castrated recipients at both collection time points in comparison to the intact recipients and non-grafted intact mouse controls. Pachytene spermatocytes were the most advanced germ cells observed in grafts recovered from castrated recipients 24 weeks post-grafting. Complete spermatogenesis, as indicated by the presence of elongated spermatids, was present only in grafts from intact recipients collected 24 weeks post-grafting. However, significant number of germ cells with DNA damage was also detected in these grafts as indicated by TUNEL assay. The complete germ cell differentiation in xenografts from intact recipients may be attributed to efficient Sertoli cell maturation. These results suggest that germ cell differentiation in buffalo testis xenograft can be completed by altering the recipient gonadal status.

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268 GERM CELL DEVELOPMENT IN EQUINE TESTIS TISSUE XENOGRAFTED INTO MICE

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab268 ◽

2005 ◽

Vol 17 (2) ◽

pp. 283 ◽

Cited By ~ 1

Author(s):

R. Turner ◽

R. Rathi ◽

A. Honaramooz ◽

W. Zeng ◽

I. Dobrinski

Keyword(s):

Chorionic Gonadotropin ◽

Germ Cells ◽

Seminal Vesicles ◽

Post Transplantation ◽

Immunodeficient Mice ◽

Donor Tissue ◽

Old Donor ◽

Pachytene Spermatocytes ◽

Donor Species ◽

Testis Tissue

Grafting of testis tissue from immature animals under the back skin of immunodeficient mice results in complete spermatogenesis, albeit with different levels of efficiency in different species. While spermatogenesis develops comparably to that in the donor species in xenografts from pigs, sheep and goats, spermatogenic differentiation is less efficient in testis tissue from cats and bulls. Testicular maturation was significantly accelerated in rhesus monkey testis grafts whereas timing was similar to that in the donor species in cats and bulls. The objective of this study was to investigate if grafting of immature horse testis tissue would result in spermatogenesis in a mouse host. Small fragments of testis tissue (about 1 mm3) from four sexually immature colts (2-week-old Standardbred, 5- and 8-month-old ponies, 10-month-old Warmblood) were grafted under the back skin of castrated male immunodeficient mice (n = 5, 5, 10 and 5 recipient mice, respectively). Histological examination of the testis xenografts was performed between 14 and 50 week post-transplantation. Weight of the seminal vesicles in the host mouse was recorded as an indicator of bioactive testosterone produced by the xenografts. At the time of grafting, the seminiferous cords of the donor testis tissue form 2-week-, 5-month- and 8-month-old colts contained only immature Sertoli cells and gonocytes. No spermatogenic differentiation occurred in xenografts from the 2-week-old colt and testosterone production was minimal. Pachytene spermatocytes were observed in testis grafts from the 5- and 8-month-old donors from 14 weeks onward. Spermatogenesis did not proceed through meiosis in grafts from the 5-month-old donor. Recipient mice carrying xenografts from the 8-month-old donor received exogenous gonadotropins (equine chorionic gonadotropin and human chorionic gonadotropin, 10 I.U./day for 2 months, beginning 14 weeks after grafting) and condensing spermatids were observed by 35 weeks after grafting. In donor tissue from the 10-month-old colt, pachytene spermatocytes were present in about 50% of tubules at the time of grafting. After 14 weeks, xenografts showed fewer differentiated germ cells than the donor tissue. However, at 35 weeks after grafting, condensing spermatids were observed, indicating that differentiated germ cells were initially lost and spermatogenesis was subsequently reinitiated. In all castrated host mice where spermatogenic differentiation occurred, the weight of the seminal vesicles was restored to pre-castration values showing that xenografts were releasing bioactive testosterone. These results indicate that horse spermatogenesis can occur in a mouse host albeit with low efficiency. Testicular maturation was not accelerated. In most cases, spermatogenesis appeared to become arrested at meiosis. The underlying mechanisms of this spermatogenic arrest require further investigation. Although equine testis xenografts produced testosterone, supplementation of exogenous gonadotropins might support post-meiotic differentiation. This work was supported by USDA 03-35203-13486.

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Germ cell fate and seminiferous tubule development in bovine testis xenografts

Reproduction ◽

10.1530/rep.1.00912 ◽

2005 ◽

Vol 130 (6) ◽

pp. 923-929 ◽

Cited By ~ 54

Author(s):

Rahul Rathi ◽

Ali Honaramooz ◽

Wenxian Zeng ◽

Stefan Schlatt ◽

Ina Dobrinski

Keyword(s):

Germ Cell ◽

Cell Fate ◽

Germ Cells ◽

Cell Number ◽

Sperm Production ◽

Continuous Increase ◽

Control Tissue ◽

Low Efficiency ◽

Pachytene Spermatocytes ◽

Testis Tissue

Spermatogenesis can occur in testis tissue from immature bulls ectopically grafted into mouse hosts; however, efficiency of sperm production is lower than in other donor species. To elucidate a possible mechanism for the impaired spermatogenesis in bovine testis xenografts, germ cell fate and xenograft development were investigated at different time points and compared with testis tissue from age-matched calves as controls. Histologically, an initial decrease in germ cell number was noticed in xenografts recovered up to 2 months post-grafting without an increase in germ cell apoptosis. From 2 months onward, the number of germ cells increased. In contrast, a continuous increase in germ cell number was seen in control tissue. Pachytene spermatocytes were observed in some grafts before 4 months, whereas in the control tissue they were not present until 5 months of age. Beyond 4 months post-grafting spermatogenesis appeared to be arrested at the pachytene spermatocyte stage in most grafts. Elongated spermatids were observed between 6 and 8 months post-grafting, similar to the controls, albeit in much lower numbers. Lumen formation started earlier in grafts compared with controls and by 6 months post-grafting tubules with extensively dilated lumen were observed. A donor effect on efficiency of spermatogenesis was also observed. These results indicate that the low efficiency of sperm production in bovine xenografts is due to an initial deficit of germ cells and impaired meiotic and post-meiotic differentiation. The characterization of spermatogenic efficiency will provide the basis to understand the control of spermatogenesis in testis grafts.

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217 PROTEIN-INDUCED TRANSDIFFERENTIATION INTO MALE GERM CELL-LIKE LINEAGE FROM CHICKEN FETAL BONE MARROW STEM CELLS

Reproduction Fertility and Development ◽

10.1071/rdv24n1ab217 ◽

2012 ◽

Vol 24 (1) ◽

pp. 220

Author(s):

J. M. Yoo ◽

J. J. Park ◽

K. Gobianand ◽

J. Y. Ji ◽

J. S. Kim ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Retinoic Acid ◽

Germ Cell ◽

Germ Cells ◽

Cultured Cells ◽

Male Germ Cell ◽

Male Germ Cells ◽

Germ Cell Differentiation ◽

Transgenic Chickens

Bone marrow (BM)-derived stem cells are capable of transdifferentiation into multilineage cells like muscle, bone, cartilage, fat and nerve cells. In this study, we investigated the capability of mesenchymal stem cells (MSC) derived from BM into germ cell differentiation in the chicken. Chicken MSCs were isolated from BM of day 20 fertilized fetal chicken with Ficoll-Paque Plus. Isolated cells were cultured in advance-DMEM (ADMEM) supplemented with 10% fetal bovine serum and antibiotics. Once confluent, cells were subcultured until five passages. The cultured cells showed fibroblast-like morphology. The cells had positive expressions of Oct4, Sox2 and Nanog. Two induction methods were conducted to examine the ability of transdifferentation into male germ cells. In group 1, MSC were cultured in ADMEM containing retinoic acid and chicken testicular extracts proteins for 10 to 15 days. In group 2, MSC were permeabilized by streptolysin O and treated with chicken testicular protein extracts. In both treatment groups, MSC were cultured in ADMEM containing retinoic acid for 10 to 15 days. We found that chicken MSC had a positive expression of pluripotent proteins such as Oct4, Sox2, Nanog and a small population of chicken MSC seem to transdifferentiate into male germ cell-like cells. These cells expressed early germ cell markers and male germ-cell-specific markers (Dazl, C-kit, Stra8 and DDX4) as analysed by reverse transcription-PCR and immunohistochemistry. These results demonstrated that chicken MSC may differentiate into male germ cells and the same might be used as a potential source of cells for production of transgenic chickens. This study was carried out with the support of Agenda Program (Project No. PJ0064692011), RDA and Republic of Korea.

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Cannabinoid Receptors Signaling in the Development, Epigenetics, and Tumours of Male Germ Cells

International Journal of Molecular Sciences ◽

10.3390/ijms21010025 ◽

2019 ◽

Vol 21 (1) ◽

pp. 25 ◽

Cited By ~ 6

Author(s):

Marco Barchi ◽

Elisa Innocenzi ◽

Teresa Giannattasio ◽

Susanna Dolci ◽

Pellegrino Rossi ◽

...

Keyword(s):

Germ Cell ◽

Germ Cells ◽

Cannabinoid Receptors ◽

Endocannabinoid System ◽

Male Reproduction ◽

Metabolizing Enzymes ◽

Male Germ Cells ◽

Testicular Tumours ◽

Germ Cell Differentiation ◽

Degradative Enzymes

Endocannabinoids are natural lipid molecules whose levels are regulated by specific biosynthetic and degradative enzymes. They bind to and activate two main cannabinoid receptors type 1 (CB1) and type 2 (CB2), and together with their metabolizing enzymes form the “endocannabinoid system” (ECS). In the last years, the relevance of endocannabinoids (eCBs) as critical modulators in various aspects of male reproduction has been pointed out. Mammalian male germ cells, from mitotic to haploid stage, have a complete ECS which is modulated during spermatogenesis. Compelling evidence indicate that in the testis an appropriate “eCBs tone”, associated to a balanced CB receptors signaling, is critical for spermatogenesis and for the formation of mature and fertilizing spermatozoa. Any alteration of this system negatively affects male reproduction, from germ cell differentiation to sperm functions, and might have also an impact on testicular tumours. Indeed, most of testicular tumours develop during early germ-cell development in which a maturation arrest is thought to be the first key event leading to malignant transformation. Considering the ever-growing number and complexity of the data on ECS, this review focuses on the role of cannabinoid receptors CB1 and CB2 signaling in male germ cells development from gonocyte up to mature spermatozoa and in the induction of epigenetic alterations in these cells which might be transmitted to the progeny. Furthermore, we present new evidence on their relevance in testicular cancer.

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Proteomic and metabolomic analyses uncover sex-specific regulatory pathways in mouse fetal germline differentiation†

Biology of Reproduction ◽

10.1093/biolre/ioaa115 ◽

2020 ◽

Vol 103 (4) ◽

pp. 717-735

Author(s):

Yohei Hayashi ◽

Masaru Mori ◽

Kaori Igarashi ◽

Keiko Tanaka ◽

Asuka Takehara ◽

...

Keyword(s):

Oxidative Phosphorylation ◽

Germ Cell ◽

Germ Cells ◽

Cell Development ◽

Nucleotide Synthesis ◽

Male Germ Cells ◽

Regulatory Pathways ◽

Germ Cell Development ◽

Germ Cell Differentiation ◽

Germline Differentiation

Abstract Regulatory mechanisms of germline differentiation have generally been explained via the function of signaling pathways, transcription factors, and epigenetic regulation; however, little is known regarding proteomic and metabolomic regulation and their contribution to germ cell development. Here, we conducted integrated proteomic and metabolomic analyses of fetal germ cells in mice on embryonic day (E)13.5 and E18.5 and demonstrate sex- and developmental stage-dependent changes in these processes. In male germ cells, RNA processing, translation, oxidative phosphorylation, and nucleotide synthesis are dominant in E13.5 and then decline until E18.5, which corresponds to the prolonged cell division and more enhanced hyper-transcription/translation in male primordial germ cells and their subsequent repression. Tricarboxylic acid cycle and one-carbon pathway are consistently upregulated in fetal male germ cells, suggesting their involvement in epigenetic changes preceding in males. Increased protein stability and oxidative phosphorylation during female germ cell differentiation suggests an upregulation of aerobic energy metabolism, which likely contributes to the proteostasis required for oocyte maturation in subsequent stages. The features elucidated in this study shed light on the unrevealed mechanisms of germ cell development.

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1 XENOGRAFTING OF ADULT MAMMALIAN TESTIS TISSUE

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab1 ◽

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).

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Mammalian male germ cells are fertile ground for expression profiling of sexual reproduction

Reproduction ◽

10.1530/rep.1.00408 ◽

2005 ◽

Vol 129 (1) ◽

pp. 1-7 ◽

Cited By ~ 44

Author(s):

Gunnar Wrobel ◽

Michael Primig

Keyword(s):

Germ Cell ◽

Germ Cells ◽

Large Scale ◽

Transcriptional Profiling ◽

Model Systems ◽

Male Germ Cells ◽

Germ Cell Differentiation ◽

Somatic Tissues ◽

Fertile Ground ◽

Expression Program

Recent large-scale transcriptional profiling experiments of mammalian spermatogenesis using rodent model systems and different types of microarrays have yielded insight into the expression program of male germ cells. These studies revealed that an astonishingly large number of loci are differentially expressed during spermatogenesis. Among them are several hundred transcripts that appear to be specific for meiotic and post-meiotic germ cells. This group includes many genes that were previously implicated in spermatogenesis and/or fertility and others that are as yet poorly characterized. Profiling experiments thus reveal candidates for regulation of spermatogenesis and fertility as well as targets for innovative contraceptives that act on gene products absent in somatic tissues. In this review, consolidated high density oligonucleotide microarray data from rodent total testis and purified germ cell samples are analyzed and their impact on our understanding of the transcriptional program governing male germ cell differentiation is discussed.

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Human Pluripotent Stem Cells in Reproductive Science—A Comparison of Protocols Used to Generate and Define Male Germ Cells from Pluripotent Stem Cells

International Journal of Molecular Sciences ◽

10.3390/ijms21031028 ◽

2020 ◽

Vol 21 (3) ◽

pp. 1028

Author(s):

Magdalena Kurek ◽

Halima Albalushi ◽

Outi Hovatta ◽

Jan-Bernd Stukenborg

Keyword(s):

Stem Cells ◽

Growth Factors ◽

Germ Cell ◽

Cell Lines ◽

Germ Cells ◽

Pluripotent Stem Cells ◽

Human Pluripotent Stem Cells ◽

Male Germ Cells ◽

Germ Cell Differentiation

Globally, fertility-related issues affect around 15% of couples. In 20%–30% of cases men are solely responsible, and they contribute in around 50% of all cases. Hence, understanding of in vivo germ-cell specification and exploring different angles of fertility preservation and infertility intervention are considered hot topics nowadays, with special focus on the use of human pluripotent stem cells (hPSCs) as a source of in vitro germ-cell generation. However, the generation of male germ cells from hPSCs can currently be considered challenging, making a judgment on the real perspective of these innovative approaches difficult. Ever since the first spontaneous germ-cell differentiation studies, using human embryonic stem cells, various strategies, including specific co-cultures, gene over-expression, and addition of growth factors, have been applied for human germ-cell derivation. In line with the variety of differentiation methods, the outcomes have ranged from early and migratory primordial germ cells up to post-meiotic spermatids. This variety of culture approaches and cell lines makes comparisons between protocols difficult. Considering the diverse strategies and outcomes, we aim in this mini-review to summarize the literature regarding in vitro derivation of human male germ cells from hPSCs, while keeping a particular focus on the culture methods, growth factors, and cell lines used.

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