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Jayanti Tokas 1, Rubina Begum 1, Shalini Jain 2 and Hariom Yadav 2 Department of Biotechnology, JMIT, Radaur, India NIDDK, National Institutes of Health,

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Presentation on theme: "Jayanti Tokas 1, Rubina Begum 1, Shalini Jain 2 and Hariom Yadav 2 Department of Biotechnology, JMIT, Radaur, India NIDDK, National Institutes of Health,"— Presentation transcript:

1 Jayanti Tokas 1, Rubina Begum 1, Shalini Jain 2 and Hariom Yadav 2 Department of Biotechnology, JMIT, Radaur, India NIDDK, National Institutes of Health, Bethesda, MD 20892, USA (A Basic Concept)

2  Spermatogenesis is the process of germ cells proliferation and differentiation within the seminiferous tubules of the testes leading to haploid, free swimming spermatozoa.  Spermatogonial stem cells (present in the seminiferous tubules) form the basis of spermatogenesis.

3  Stem cells are characterized according to the tissue from which the cells are derived. # Embryonic stem cells # Adult stem cells  Amongst the adult stem cells, the two best defined systems are : *Spermatogenesis (Spermatogonial stem cells, SSC) *Haematopoesis (Haematopoeitic stem cells, HSC)

4 Spermatogonial Stem cells (SSCs) (defined by their functions) Self-renewal - ability to go through numerous cell divisions while maintaining the undifferentiated state. Self-renewal - ability to go through numerous cell divisions while maintaining the undifferentiated state. Multipotent - capacity to differentiate into any type of mature cell. Multipotent - capacity to differentiate into any type of mature cell.

5 SSCs are the only adult stem cells that transmit genetic information to next generation SSCs are the only adult stem cells that transmit genetic information to next generation These are the eternal germ cells present from birth to death These are the eternal germ cells present from birth to death SSC self renew and produce daughter cells that differentiate into spermatozoa. SSC self renew and produce daughter cells that differentiate into spermatozoa. Reside within the basal layer of seminiferous tubules of the testes. Reside within the basal layer of seminiferous tubules of the testes. Maintain spermatogenesis throughout life in males by proliferation and differentiation. Maintain spermatogenesis throughout life in males by proliferation and differentiation.

6 Fate of the spermatogonial stem cells Type A spermatogonia Type B spermatogoniaCell death RenewalDifferentiation Apoptosis

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8 Extra embryonic ectoderm Epiblast Visceral endoderm 1. Migration of primordial germ cells to allantois region

9 2. Migration of PGCs endoderm Allantois PGCs

10 3. Migration of PGCs into gonad Prenatal – PGCs associate with sex cords and secondary sex cords become seminiferous tubules.

11 Primordial Germ Cells differentiate to become gonocytes Proliferate Mitotic arrest until birth (depending on species) undergo Rodents – around birth Bulls – 4-8 weeks of age Boars – 5-15 days of age Humans – 2 yr Gonocytes can differentiate into spermatogonial stem cells or spermatogonia

12 After birth gonocytes # Resume mitosis # Migrate to basement membrane # Differentiate into type A o spermatogonia * Spermatogonial stem cells originate from PGC at 7.5 days p.c.

13 Spermatogonial stem cells present in the testis Niche - subset of tissue cells and extracellular substrates that can indefinitely house one or more stem cells and control their self-renewal and progeny production in vivo.

14 Identification of spermatogonial stem cells Until 1977 – Isolation Until 1977 – Isolation Bovine serum albumin gradient and velocity sedimentation (90% purity) Bovine serum albumin gradient and velocity sedimentation (90% purity) Kit ligand/c-Kit receptor Kit ligand/c-Kit receptor Growth factor- produced by Sertoli cells Growth factor- produced by Sertoli cells Regulate the growth of the spermatogonia Regulate the growth of the spermatogonia Used as marker for A spermatogonia Used as marker for A spermatogonia

15 Stages of cell division can be used to distinguish between stem cells and differentiating cells Stages of cell division can be used to distinguish between stem cells and differentiating cells SSCs activities can be identified by the formation of colonies SSCs activities can be identified by the formation of colonies Surface phenotype for identification Surface phenotype for identification Thy-1- unique marker for mouse and rat SSC Thy-1- unique marker for mouse and rat SSC Surface phenotype - MHC-1 Thy-1 c-Kit, av-integrin –these markers used for identification of SSC (approx 1 SC in 15 total cells) using FACS or MACS Surface phenotype - MHC-1 Thy-1 c-Kit, av-integrin –these markers used for identification of SSC (approx 1 SC in 15 total cells) using FACS or MACS

16 Thy -1 β-1 and α6 integrin Stra 8 CD9 antigen c-Kit receptor Spermatogonial stem cells share some, but not all phenotypic and functional characteristics with other stem cells. Markers of spermatogonial stem cells

17 Self-renewal Self-renewal maintains spermatogenesis Self-renewal maintains spermatogenesis Factors regulating the self-renewal and differentiation Factors regulating the self-renewal and differentiation Intrinsic gene expression Intrinsic gene expression Extrinsic signals (soluble factors) Extrinsic signals (soluble factors) Adhesion of molecules Adhesion of molecules Regulatory mechanism remains elusive

18 Spermatogonial control mechanism Sertoli cells limits the expansion of SSC population Sertoli cells limits the expansion of SSC population Hormones do not influence the expansion of spermatogonial cells Hormones do not influence the expansion of spermatogonial cells Regulated automatically or genetically Regulated automatically or genetically Stem Cell Factor and its receptor c-kit regulate spermatogonial development Stem Cell Factor and its receptor c-kit regulate spermatogonial development

19 SSCs maintained in culture without losing their proliferation and differentiation potential. (Nagano et al., 1998) (Nagano et al., 1998) SSCs maintained in co-culture with sertoli cells without loosing the ability to replicate their DNA. (Van Der Wee et al., 2001) (Van Der Wee et al., 2001) SCF and GM-CSF enhance the survival of porcine type A SSCs (Dirami et al., 1999) SSCs can expand in complete absence of serum or somatic feeder cells in vitro. (Kanatsu-Shinohara et al., 2005) (Kanatsu-Shinohara et al., 2005) SSCs can undergo anchorage-independent, self-renewal division in vitro. (Kanatsu-Shinohara et al., 2006) (Kanatsu-Shinohara et al., 2006)Culture

20 Cryopreservation of SSCs Successful transplantation after freezing the donor tissue for 156 days Successful transplantation after freezing the donor tissue for 156 days (Avarbock and colleagues, 1996 ) (Avarbock and colleagues, 1996 ) Frozen thawed bovine SSCs survive cryopreservation maintained during co-culture, maintenance is influenced by GDNF. Frozen thawed bovine SSCs survive cryopreservation maintained during co-culture, maintenance is influenced by GDNF. Donor testis cells isolated from different species frozen up to 96 days at ­196 °C were able to generate spermatogenesis in recipient seminiferous tubules. (Avarbock et al., 1996) Donor testis cells isolated from different species frozen up to 96 days at ­196 °C were able to generate spermatogenesis in recipient seminiferous tubules. (Avarbock et al., 1996) Cryopreserved testis cells of dogs and rabbits are capable of colonizing the recipient mouse testis. Cryopreserved testis cells of dogs and rabbits are capable of colonizing the recipient mouse testis. (Dobrinski et al.,1999) (Dobrinski et al.,1999)  Bovine type A spermatogonia survived after 2-4 months of cryopreservation. (Izadyar et al.,2002) (Izadyar et al.,2002)

21 Stage specific expression of Tsp57 mRNA indicates that it has very specific role during the haploid phase of spermatogenesis. (Kim et al., 2004) Pin-1 is required to regulate proliferation and cell fate of undifferentiated spermatogonia in the adult mouse testis. (Atchison et al., 2003) Pin-1 is required to regulate proliferation and cell fate of undifferentiated spermatogonia in the adult mouse testis. (Atchison et al., 2003) Plzf null mice share similar defects in sperm production that are due to an inability of spermatogonial stem cells to self renew. (Kotaja et al., 2004) Dazl expression is predominant in the primary spermatocytes and weak in spermatogonia. (Lin et al., 2001) Maintenance of spermatogenesis requires TAF4b and a requisite for fertility in mice. (Falender et al., 2005) Bcl6b is a critical molecule for SSC function and also an important component in maintaining normal SSC biology and spermatogenesis in vivo. Genes responsible for SSCs maintenance

22 Transplantation of SSCs Brinster and Zimmerman in Ist time Brinster and Zimmerman in Ist time Cellular differentiation was also started after injecting the cellular suspension Cellular differentiation was also started after injecting the cellular suspension They found that donor- derived spermatogonia were responsible for producing offspring They found that donor- derived spermatogonia were responsible for producing offspring Transplantation between greater distant animals has been less successful. This is likely due to failed spermatogonia and sertoli cell structural association and other functional interactions. Transplantation between greater distant animals has been less successful. This is likely due to failed spermatogonia and sertoli cell structural association and other functional interactions. As a result As a result Production of morphologically defective spermatozoa Production of morphologically defective spermatozoa Successful spermatogenesis obtained following human-to-rat and mouse transplantation (Sofikitis et al, 1999) Successful spermatogenesis obtained following human-to-rat and mouse transplantation (Sofikitis et al, 1999) Human to immunodeficient mouse testicular tissue transplantation, no evidence of donor tissue survival.( Reis et al, 2000) Human to immunodeficient mouse testicular tissue transplantation, no evidence of donor tissue survival. ( Reis et al, 2000)

23  Human spermatogonia in mouse survived up to 6 months but no meiotic activity was found in donor tissues (Nagano et al, 2002)  Mouse seminiferous tubules provide a suitable environment for germ cells from distant species to interact with supporting cells and associate with basement membrane. (Dobrinski et al., 1999)  Transplantation of hamster germ cells into mouse testes resulted in donor-derived spermatogenesis. (Ogawa et al., 1999)  Successful transplantation of bovine type A spermatogonia in recipient bulls resulting in full spermatogenesis after autologus transplantation. (F.Izadyar et al., 2003)

24  To date, donor–derived spermatogenesis has been primarily limited to similar species  Mouse-to–mouse, Rat-to-mouse, Hamster-to- mouse  It has been said that evolutionary distance is primarily responsible for the failure of transplantation  Success was obtained in similar species only

25 Spermatogonial stem cells transplantation would act as a wonderful tool to Study the early male germ cell development. Study the surface markers. Study the genes & factors involved in regulation of proliferation and differentiation of spermatogonial stem cells. Preservation of germ line in valuable males. Transgenic animals.

26 Practical implications 1. Spermatogenic process can be reinitiated in the patients those who have lost their spermatogonial cells during the treatment for such diseases. 2. Transplantation of spermatogonial stem cells in the recipient’s seminiferous tubules for reinitiation of spermatozoa production in injury and other cytotoxic damages 3. In-vitro spermatogenesis 4. Production of transgenic animals (more easy than the ESC technique) 5. Development of male contraception 6. Cryopreservation of reserve sperms and combined with artificial reproduction techniques. 7. Animal conservation 8. Multipotent adult germline stem cells can be used for individual cell based therapy without the ethical and immunological problems associated with human embryonic stem cells 9. Acts as precursor in case of natural depletion 10. Alternative strategy for fertility preservation

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