(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
 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.
 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)
Spermatogonial Stem cells (SSCs)
(defined by their functions)

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Self-renewal - ability to go through numerous cell
divisions while maintaining the undifferentiated
state.
Multipotent - capacity to differentiate into any
type of mature cell.

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.
Fate of the spermatogonial stem cells
Type A spermatogonia
Renewal
Type A spermatogonia
Differentiation
Type B spermatogonia
Apoptosis
Cell death
1. Migration of primordial germ cells to
allantois region
Extra embryonic
ectoderm
Epiblast
Visceral endoderm
2. Migration of PGCs endoderm
Allantois
PGCs
3. Migration of PGCs into gonad
Prenatal – PGCs associate with sex cords and secondary sex
cords become seminiferous tubules.
Primordial Germ Cells differentiate to become gonocytes
Proliferate
undergo
Mitotic arrest until birth
(depending on species)
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
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.
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.
Identification of spermatogonial stem
cells

Until 1977 – Isolation

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Bovine serum albumin gradient and velocity
sedimentation (90% purity)
1990- Kit ligand/c-Kit receptor
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Growth factor- produced by Sertoli cells
Regulate the growth of the spermatogonia
Used as marker for A spermatogonia

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

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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
Markers of spermatogonial stem cells
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.
Self-renewal

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Self-renewal maintains spermatogenesis
Factors regulating the self-renewal
differentiation

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Intrinsic gene expression
Extrinsic signals (soluble factors)
Adhesion of molecules
Regulatory mechanism remains elusive
and
Spermatogonial control mechanism
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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
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)
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)


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.
Transplantation of SSCs
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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)
To date, donor–derived spermatogenesis has
been primarily limited to similar species
 Mouse-to–mouse, Rat-to-mouse, Hamster-tomouse
 It has been said that evolutionary distance is
primarily responsible for the failure of
transplantation
 Success was obtained in similar species only

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.
Practical implications
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Spermatogenic process can be reinitiated in the patients those who have lost
their spermatogonial cells during the treatment for such diseases.
Transplantation of spermatogonial stem cells in the recipient’s seminiferous
tubules for reinitiation of spermatozoa production in injury and other
cytotoxic damages
In-vitro spermatogenesis
Production of transgenic animals (more easy than the ESC technique)
Development of male contraception
Cryopreservation of reserve sperms and combined with artificial
reproduction techniques.
Animal conservation
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
Acts as precursor in case of natural depletion
Alternative strategy for fertility preservation