Falana B.A.

Department of Anatomy, College of Medicine, University of Lagos.

(A Basic Concept)

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)

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)Spermatogonial Stem cells (SSCs) (defined by their functions)(defined by their functions)

Self-renewalSelf-renewal - ability to go through numerous cell - ability to go through numerous cell divisions while maintaining the undifferentiated divisions while maintaining the undifferentiated state. state.

MultipotentMultipotent - capacity to differentiate into any - capacity to differentiate into any type of mature cell. type of mature cell.

SSCs are the only adult stem cells that transmit genetic SSCs are the only adult stem cells that transmit genetic information to next generationinformation to next generation

These are the eternal germ cells present from birth to These are the eternal germ cells present from birth to deathdeath

SSC self renew and produce daughter cells that SSC self renew and produce daughter cells that differentiate into spermatozoa.differentiate into spermatozoa.

Reside within the basal layer of seminiferous tubules of Reside within the basal layer of seminiferous tubules of the testes.the testes.

Maintain spermatogenesis throughout life in males by Maintain spermatogenesis throughout life in males by proliferation and differentiation.proliferation and differentiation.

Fate of the spermatogonial stem cellsFate of the spermatogonial stem cells

Type A spermatogonia

Type A spermatogonia Type B spermatogonia Cell death

Renewal Differentiation Apoptosis

Extra embryonic ectoderm

Epiblast

Visceral endoderm

1. Migration of primordial germ cells to allantois region

2. Migration of PGCs endoderm2. Migration of PGCs endoderm

Allantois

PGCs

3. Migration of PGCs into gonad3. 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

Mitotic arrest until birth

(depending on species)

undergo

Rodents – around birthBulls – 4-8 weeks of ageBoars – 5-15 days of ageHumans – 2 yr

Gonocytes can differentiate into spermatogonial stem cellsor 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 Identification of spermatogonial stem cellscells

Until 1977Until 1977 – – IsolationIsolation Bovine serum albumin gradient and velocity Bovine serum albumin gradient and velocity

sedimentation (90% purity)sedimentation (90% purity) 1990-1990- Kit ligand/c-Kit receptorKit ligand/c-Kit receptor

Growth factor- produced by Sertoli cellsGrowth factor- produced by Sertoli cells Regulate the growth of the spermatogoniaRegulate the growth of the spermatogonia Used as marker forUsed as marker for A spermatogonia A spermatogonia

Stages of Stages of cell divisioncell division can be used to distinguish can be used to distinguish between stem cells and differentiating cellsbetween stem cells and differentiating cells

SSCs activities can be identified by the formation SSCs activities can be identified by the formation of of coloniescolonies

Surface phenotype for identification Surface phenotype for identification Thy-1-Thy-1- unique marker for mouse and rat SSC unique marker for mouse and rat SSC

Surface phenotype - Surface phenotype - MHC-1 Thy-1 c-Kit, av-integrinMHC-1 Thy-1 c-Kit, av-integrin –these –these markers used for identification of SSC (approx 1 SC in 15 markers used for identification of SSC (approx 1 SC in 15 total cells) using total cells) using FACSFACS or or MACSMACS

Thy -1Thy -1

ββ--1 and 1 and αα6 integrin6 integrin

Stra 8Stra 8

CD9 antigen CD9 antigen

c-Kit receptor c-Kit receptor

Spermatogonial stem cells share some, but not all Spermatogonial stem cells share some, but not all phenotypic and functional characteristics with other stem phenotypic and functional characteristics with other stem

cells.cells.

Markers of spermatogonial stem cellsMarkers of spermatogonial stem cells

Self-renewalSelf-renewal

Self-renewal maintains spermatogenesisSelf-renewal maintains spermatogenesis Factors regulating the self-renewal and Factors regulating the self-renewal and

differentiationdifferentiation Intrinsic gene expression Intrinsic gene expression Extrinsic signals (soluble factors)Extrinsic signals (soluble factors) Adhesion of moleculesAdhesion of molecules

Regulatory mechanism remains elusive Regulatory mechanism remains elusive

Spermatogonial control mechanismSpermatogonial control mechanism

Sertoli cells limits the expansion of SSC Sertoli cells limits the expansion of SSC populationpopulation

Hormones do not influence the expansion of Hormones do not influence the expansion of spermatogonial cellsspermatogonial cells

Regulated automatically or geneticallyRegulated automatically or genetically Stem Cell Factor and its receptor c-kit regulate Stem Cell Factor and its receptor c-kit regulate

spermatogonial developmentspermatogonial development

SSCs maintained in culture without losing their proliferation SSCs maintained in culture without losing their proliferation and differentiation potential.and differentiation potential.

(Nagano (Nagano et al.et al., 1998), 1998)

SSCs maintained in co-culture with sertoli cells without loosing SSCs maintained in co-culture with sertoli cells without loosing the ability to replicate their DNA.the ability to replicate their DNA.

(Van Der Wee (Van Der Wee et al.et al., 2001), 2001)

SCF and GM-CSF enhance the survival of porcine type A SSCs SCF and GM-CSF enhance the survival of porcine type A SSCs (Dirami (Dirami et al.et al., 1999), 1999)

SSCs can expand in complete absence of serum or somatic SSCs can expand in complete absence of serum or somatic feeder cells in vitro.feeder cells in vitro.

(Kanatsu-Shinohara (Kanatsu-Shinohara et al.et al., 2005), 2005)SSCs can undergo anchorage-independent, self-renewal SSCs can undergo anchorage-independent, self-renewal division in vitro.division in vitro.

(Kanatsu-Shinohara (Kanatsu-Shinohara et al.et al., 2006), 2006)

CultureCulture

Cryopreservation of SSCsCryopreservation of SSCs Successful transplantation after freezing the donor tissue for 156 daysSuccessful 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 Frozen thawed bovine SSCs survive cryopreservation maintained during

co-culture, maintenance is influenced by GDNF. co-culture, maintenance is influenced by GDNF.

Donor testis cells isolated from different species frozen up to 96 days at Donor testis cells isolated from different species frozen up to 96 days at 196 °C were able to generate spermatogenesis in recipient seminiferous 196 °C were able to generate spermatogenesis in recipient seminiferous tubules. tubules. (Avarbock (Avarbock et al.et al., 1996), 1996)

Cryopreserved testis cells of dogs and rabbits are capable of colonizing Cryopreserved testis cells of dogs and rabbits are capable of colonizing the recipient mouse testis.the recipient mouse testis.

(Dobrinski (Dobrinski et al.,et al.,1999)1999) Bovine type A spermatogonia survived after 2-4 months of Bovine type A spermatogonia survived after 2-4 months of

cryopreservation.cryopreservation.

(Izadyar (Izadyar et al.,et al.,2002)2002)

Stage specific expression of Stage specific expression of TspTsp57 mRNA indicates that it has very specific role 57 mRNA indicates that it has very specific role during the haploid phase of spermatogenesis. during the haploid phase of spermatogenesis. (Kim (Kim et al.et al., 2004), 2004) PinPin-1 is required to regulate proliferation and cell fate of undifferentiated -1 is required to regulate proliferation and cell fate of undifferentiated spermatogonia in the adult mouse testis. spermatogonia in the adult mouse testis.

(Atchison (Atchison et al.et al., 2003), 2003) PlzfPlzf null mice share similar defects in sperm production that are due to an null mice share similar defects in sperm production that are due to an inability of spermatogonial stem cells to self renew. inability of spermatogonial stem cells to self renew.

(Kotaja (Kotaja et al.et al., 2004), 2004)DazlDazl expression is predominant in the primary spermatocytes and weak in expression is predominant in the primary spermatocytes and weak in spermatogonia. spermatogonia. (Lin (Lin et al.et al., 2001), 2001)Maintenance of spermatogenesis requires Maintenance of spermatogenesis requires TAFTAF4b and a requisite for fertility in 4b and a requisite for fertility in mice. mice.

(Falender (Falender et al.et al., 2005), 2005) Bcl6bBcl6b is a critical molecule for SSC function and also an important component is a critical molecule for SSC function and also an important component in maintaining normal SSC biology and spermatogenesis in vivo.in maintaining normal SSC biology and spermatogenesis in vivo.

Genes responsible for SSCs maintenanceGenes responsible for SSCs maintenance

Transplantation of SSCsTransplantation of SSCs

Brinster and Zimmerman in 1994- Ist timeBrinster and Zimmerman in 1994- Ist time Cellular differentiation was also started after injecting the cellular suspensionCellular differentiation was also started after injecting the cellular suspension They found that donor- derived spermatogonia were responsible for producing They found that donor- derived spermatogonia were responsible for producing

offspringoffspring Transplantation between greater distant animals has been Transplantation between greater distant animals has been lessless successful. successful.

This is likely due to failed spermatogonia and sertoli cell structural This is likely due to failed spermatogonia and sertoli cell structural association and other functional interactions. association and other functional interactions.

As a result As a result Production of morphologically defective spermatozoaProduction of morphologically defective spermatozoa Successful spermatogenesis obtained following human-to-rat and mouse Successful spermatogenesis obtained following human-to-rat and mouse

transplantation transplantation (Sofikitis (Sofikitis et alet al, , 1999)1999)

Human to immunodeficient mouse testicular tissue transplantation, Human to immunodeficient mouse testicular tissue transplantation, no no evidence of donor tissue survivalevidence of donor tissue survival.. (( Reis Reis et al,et al, 2000)2000)

Human spermatogonia in mouse survived up to 6 months Human spermatogonia in mouse survived up to 6 months but but no meiotic activity was found in donor tissues no meiotic activity was found in donor tissues

(Nagano et al, 2002)(Nagano et al, 2002) Mouse seminiferous tubules provide a suitable environment Mouse seminiferous tubules provide a suitable environment

for germ cells from distant species to interact with supporting for germ cells from distant species to interact with supporting cells and associate with basement membrane. cells and associate with basement membrane. (Dobrinski (Dobrinski et al., et al., 1999)1999)

Transplantation of hamster germ cells into mouse testes Transplantation of hamster germ cells into mouse testes resulted in donor-derived spermatogenesis. resulted in donor-derived spermatogenesis.

(Ogawa (Ogawa et al.et al., 1999), 1999)

Successful transplantation of bovine type A spermatogonia in Successful transplantation of bovine type A spermatogonia in recipient bulls resulting in full spermatogenesis after recipient bulls resulting in full spermatogenesis after autologus transplantation. autologus transplantation.

(F.Izadyar et al., 2003)(F.Izadyar et al., 2003)

To date, donor–derived spermatogenesis has To date, donor–derived spermatogenesis has been primarily limited to similar speciesbeen primarily limited to similar species

Mouse-to–mouse, Rat-to-mouse, Hamster-to-Mouse-to–mouse, Rat-to-mouse, Hamster-to-mousemouse

It has been said that evolutionary distance is It has been said that evolutionary distance is primarily responsible for the failure of primarily responsible for the failure of

transplantationtransplantation Success was obtained in similar species onlySuccess was obtained in similar species only

Spermatogonial stem cells transplantation would act as Spermatogonial stem cells transplantation would act as a wonderful tool toa wonderful tool to

Study the early male germ cell development.Study the early male germ cell development.Study the surface markers.Study the surface markers.Study the genes & factors involved in regulation of Study the genes & factors involved in regulation of proliferation and differentiation of spermatogonial proliferation and differentiation of spermatogonial stem cells.stem cells.Preservation of germ line in valuable males.Preservation of germ line in valuable males.Transgenic animals.Transgenic animals.

PracticalPractical implicationsimplications

1.1. Spermatogenic process can be reinitiated in the patients those who have Spermatogenic process can be reinitiated in the patients those who have lost their spermatogonial cells during the treatment for such diseases. lost their spermatogonial cells during the treatment for such diseases.

2.2. Transplantation of spermatogonial stem cells in the recipient’s Transplantation of spermatogonial stem cells in the recipient’s seminiferous tubules for reinitiation of spermatozoa production in injury seminiferous tubules for reinitiation of spermatozoa production in injury and other cytotoxic damagesand other cytotoxic damages

3.3. In-vitro spermatogenesisIn-vitro spermatogenesis4.4. Production of transgenic animals (more easy than the ESC technique)Production of transgenic animals (more easy than the ESC technique)5.5. Development of male contraception Development of male contraception 6.6. Cryopreservation of reserve sperms and combined with artificial Cryopreservation of reserve sperms and combined with artificial

reproduction techniques.reproduction techniques.7.7. Animal conservationAnimal conservation8.8. Multipotent adult germline stem cells can be used for individual cell based Multipotent adult germline stem cells can be used for individual cell based

therapy without the ethical and immunological problems associated with therapy without the ethical and immunological problems associated with human embryonic stem cellshuman embryonic stem cells

9.9. Acts as precursor in case of natural depletionActs as precursor in case of natural depletion10.10. Alternative strategy for fertility preservationAlternative strategy for fertility preservation