Spermatogonial stem cells (A Basic Concept)

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Spermatogonial stem cells
(A Basic Concept)
Jayanti Tokas1, Rubina Begum1, Shalini Jain2 and
Hariom Yadav2 Department of Biotechnology, JMIT,
Radaur, India NIDDK, National Institutes of
Health, Bethesda, MD 20892, USA
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• 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.

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• 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)

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

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• 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
• SSC self renew and produce daughter cells that
  differentiate into spermatozoa.
• Reside within the basal layer of seminiferous
  tubules of the testes.
• Maintain spermatogenesis throughout life in males
  by proliferation and differentiation.

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Fate of the spermatogonial stem cells
Type A spermatogonia
Renewal
Differentiation
Apoptosis
Type B spermatogonia
Cell death
Type A spermatogonia
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ESTABLISHMENT OF MALE GERM LINE
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1. Migration of primordial germ cells to
allantois region
Extra embryonic ectoderm
Epiblast
Visceral endoderm
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2. Migration of PGCs endoderm
Allantois
PGCs
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3. Migration of PGCs into gonad
Prenatal PGCs associate with sex cords and
secondary sex cords become seminiferous tubules.
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• 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
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After birth gonocytes
Resume mitosis Migrate to
basement membrane
Differentiate into type Ao spermatogonia
Spermatogonial stem cells originate from PGC at
7.5 days p.c.
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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.
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Identification of spermatogonial stem cells

• Until 1977 Isolation
• Bovine serum albumin gradient and velocity
  sedimentation (90 purity)
• 1990- Kit ligand/c-Kit receptor
• Growth factor- produced by Sertoli cells
• Regulate the growth of the spermatogonia
• Used as marker for A spermatogonia

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• 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
• Surface phenotype for identification
• 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

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Markers of spermatogonial stem cells

• Thy -1
• 
  
  
  
• ß-1 and a6 integrin
• Stra 8
• CD9 antigen
• c-Kit receptor
• Spermatogonial stem cells share some, but not all
  phenotypic and functional characteristics with
  other stem cells.
• 
  

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Self-renewal

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

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Spermatogonial control mechanism

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

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Culture

• SSCs maintained in culture without losing their
  proliferation and differentiation potential.
• 
  (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)
• 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)
• SSCs can undergo anchorage-independent,
  self-renewal division in vitro.
• 
  (Kanatsu-Shinohara et al., 2006)

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Cryopreservation of SSCs

• Successful transplantation after freezing the
  donor tissue for 156 days
• (Avarbock and colleagues, 1996 )
• 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)
• Cryopreserved testis cells of dogs and rabbits
  are capable of colonizing the recipient mouse
  testis.
• 
  (Dobrinski et al.,1999)
• Bovine type A spermatogonia survived after 2-4
  months of cryopreservation.
• 
  (Izadyar et al.,2002)
  

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Genes responsible for SSCs maintenance

• 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)
• 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.

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Transplantation of SSCs

• Brinster and Zimmerman in 1994- Ist time
• Cellular differentiation was also started after
  injecting the cellular suspension
• 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.
• As a result
• Production of morphologically defective
  spermatozoa
• 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)

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• 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)

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• To date, donorderived spermatogenesis has been
  primarily limited to similar species
• Mouse-tomouse, 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

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• 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.

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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 recipients 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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Thank You