Efficacy of Ficus carica leaf extract on morphological and molecular behavior of mice germ stem cells

Infertility is one of the most prevalent health disorders in reproductive-age males and females. Ficus carica (Fc), an herbal plant, has been used traditionally for the treatment of different diseases such as infertility especially in Iranian folk medicine. This study examined the effects of Fc leaf extract on the proliferation of mice spermatogonial stem cells (SSCs). Phenolic, flavonoid content, major polyphenolic compounds and antioxidant activity of the extract was evaluated respectively by Folin-Ciocateu, aluminum chloride, HPLC and the FRAP and DPPH methods. Testicular cells of neonate mice were extracted and their identity was confirmed using cytokeratin for Sertoli and Oct-4, CDHI and PLZF for SSCs. Effects of Fc (0.0875, 0.175, 0.35, 0.71 and 1.42 mg/ml) was evaluated at third, 7th, 9th and 14th days of culture by colony assay. The expression of the Mvh, GFRα1 and Oct-4 genes and the viability and proliferation of cultured cells was assessed at the end of the culture period. The extract has a rich phenolic and flavonoid content such as Rutin, Psoralen, Bergapten and Caffeoylmalic acid using HPLC analysis. It also had a potent reducing and radical scavenging activity. Morphology of colonies was similar in all groups. Higher viability, proliferation, colony number and diameter of SSCs was seen in the presence of Fc leaf extract in a dosedependent manner so that higher number and diameter of colonies were observed in two higher doses of 0.71 and 1.42 mg/ml, separately for each time point relative to other groups. The Mvh, Oct-4 and GFRα1 genes expression had no significant differences between groups. It seems that Fc leaf extract not only had no any cytotoxic effects on the viability and proliferation of SSCs but also support their stemness state. So, this culture system can be employed for enrichment of germ stem cells for use in clinical applications.(AU)

The role of Rho-associated kinase inhibitor, Y-27632 on primary culture of ovine spermatogonial stem cells

Abstract The access to sufficient numbers of spermatogonial stem cells (SSCs) is a prerequisite for the study of their regulation and further biomanipulation. Rho kinase (ROCK) belongs to a family of serine/threonine kinases and involves in a wide range of fundamental cellular functions. The aim of the present study was to study the effect of ROCK inhibitor, Y-27632 (0.1-40 µM), during the primary culture of ovine SSCs. SSCs were collected from 3-5-month-old's lamb testes. The viability of SSCs, the apoptosis assay of SSCs, the intracellular reactive oxygen species (ROS) analysis, and the SSCs markers and apoptosis-related gene expressions were detected by MTT reduction assay, Annexin V-FITC/ Propidium Iodide (PI) dual staining, flow cytometry and real-time-PCR studies, respectively. Morphological analyses indicated that the 5-10 µM Y-27632 had an optimal effect on the number of presumptive SSCs colonies and the area covered by them after a 10 days culture. The cell viability, apoptosis and necrosis of SSCs after 10 days' culture were not affected in comparison with the control group, and the 20 µM of Y-27632 resulted in significantly decreased cell viability (P<0.05) and an increased necrosis of cells. On day 10 after culture, the expression of P53 was decreased with an increase from 0 to 10 µM in the Y-27632 dose. In the 20 µM Y-27632 group, the expressions of P53 and Bax were higher and the Bcl-2 was lower than other groups and these values were significantly different from 5 and 10 µM Y-27632 groups (P<0.05). The level of intracellular ROS was decreased with an increase in the Y-27632 dose from 5 to 20 µM in comparison with the control group. In conclusion, the present study demonstrated that Y-27632 at a concentration of 5-10 µM provided optimal culture conditions for the primary culture of ovine SSCs.

The role of Rho-associated kinase inhibitor, Y-27632 on primary culture of ovine spermatogonial stem cells

The access to sufficient numbers of spermatogonial stem cells (SSCs) is a prerequisite for the study of their regulation and further biomanipulation. Rho kinase (ROCK) belongs to a family of serine/threonine kinases and involves in a wide range of fundamental cellular functions. The aim of the present study was to study the effect of ROCK inhibitor, Y-27632 (0.1-40 µM), during the primary culture of ovine SSCs. SSCs were collected from 3-5-month-olds lamb testes. The viability of SSCs, the apoptosis assay of SSCs, the intracellular reactive oxygen species (ROS) analysis, and the SSCs markers and apoptosis-related gene expressions were detected by MTT reduction assay, Annexin V–FITC/ Propidium Iodide (PI) dual staining, flow cytometry and real-time-PCR studies, respectively. Morphological analyses indicated that the 5-10 µM Y-27632 had an optimal effect on the number of presumptive SSCs colonies and the area covered by them after a 10 days culture. The cell viability, apoptosis and necrosis of SSCs after 10 days culture were not affected in comparison with the control group, and the 20 µM of Y-27632 resulted in significantly decreased cell viability (P<0.05) and an increased necrosis of cells. On day 10 after culture, the expression of P53 was decreased with an increase from 0 to 10 µM in the Y-27632 dose. In the 20 µM Y-27632 group, the expressions of P53 and Bax were higher and the Bcl-2 was lower than other groups and these values were significantly different from 5 and 10 µM Y-27632 groups (P<0.05). The level of intracellular ROS was decreased with an increase in the Y-27632 dose from 5 to 20 µM in comparison with the control group. In conclusion, the present study demonstrated that Y-27632 at a concentration of 5-10 µM provided optimal culture conditions for the primary culture of ovine SSCs.(AU)

Bioengenharia: transplante de células-tronco espermatogoniais em mamíferos / Bioengineering: spermatogonial stem cells transplantation in mammals

O transplante de espermatogônias tronco (SSCs, do inglês Spermatogonial Stem Cell) é uma biotecnologia que consiste na transferência de células tronco testiculares de um doador fértil para um receptor cuja espermatogênese endógena foi suprimida. Essa técnica pode ser aplicada para a produção de machos que gerem uma progênie com características genotípicas do doador selecionado. Especialmente na bovinocultura, tanto de leite como de corte, o transplante de SSCs tem o potencial de substituir a inseminação artificial (IA). Pode-se também, colocar SSCs de um mesmo doador (de genética superior) em mais de um receptor o que aumentaria o número de filhos desse doador. Além disso, possui outras aplicações como a restauração da fertilidade em homens após o tratamento de câncer, conservação de espécies ameaçadas de extinção e tratamento de causas específicas de infertilidade. Assim, com esta revisão tem-se como propósito discorrer acerca de uma biotecnologia da reprodução que permitirá a propagação do valor genético de doadores de sêmen considerados de alto valor zootécnico.

Spermatogonial Stem Cell (SSCs) transplantation is a biotechnology that consists in the transfer of testicular stem cells from a fertile donor to a recipient whose endogenous spermatogenesis has been depleted. This technique can be applied to the production of males that generate a progeny with genotypic characteristics of the selected donor. Especially in beef and dairy cattle, SSCs transplantation has the potential to replace artificial insemination (AI). In addition, it has other applications such as restoring fertility in human species after cancer treatment, conserving endangered species and treating specific causes of infertility. Thus, this aim of this review is to discuss the perspectives of reproductive biotechnology that allows the propagation of the genetic material of high pedigree males.

Bioengenharia: transplante de células-tronco espermatogoniais em mamíferos / Bioengineering: spermatogonial stem cells transplantation in mammals

O transplante de espermatogônias tronco (SSCs, do inglês Spermatogonial Stem Cell) é uma biotecnologia que consiste na transferência de células tronco testiculares de um doador fértil para um receptor cuja espermatogênese endógena foi suprimida. Essa técnica pode ser aplicada para a produção de machos que gerem uma progênie com características genotípicas do doador selecionado. Especialmente na bovinocultura, tanto de leite como de corte, o transplante de SSCs tem o potencial de substituir a inseminação artificial (IA). Pode-se também, colocar SSCs de um mesmo doador (de genética superior) em mais de um receptor o que aumentaria o número de filhos desse doador. Além disso, possui outras aplicações como a restauração da fertilidade em homens após o tratamento de câncer, conservação de espécies ameaçadas de extinção e tratamento de causas específicas de infertilidade. Assim, com esta revisão tem-se como propósito discorrer acerca de uma biotecnologia da reprodução que permitirá a propagação do valor genético de doadores de sêmen considerados de alto valor zootécnico.(AU)

Spermatogonial Stem Cell (SSCs) transplantation is a biotechnology that consists in the transfer of testicular stem cells from a fertile donor to a recipient whose endogenous spermatogenesis has been depleted. This technique can be applied to the production of males that generate a progeny with genotypic characteristics of the selected donor. Especially in beef and dairy cattle, SSCs transplantation has the potential to replace artificial insemination (AI). In addition, it has other applications such as restoring fertility in human species after cancer treatment, conserving endangered species and treating specific causes of infertility. Thus, this aim of this review is to discuss the perspectives of reproductive biotechnology that allows the propagation of the genetic material of high pedigree males.(AU)

Spermatogonial stem cell survival in ram lambs following busulfan treatment

To clarify the effect of busulfan on the depletion of spermatogonial stem cells (SSCs) from shal rams testis, in the first experiment, lambs were treated by intraperitoneal injection with 4 mg/kg busulfan. In the second experiment, different concentrations of busulfan (1, 2 and 4 mg/kg) were injected directly into both sides of the left testis. The testes of 8 lambs were collected by standard castration procedure for histological analysis five weeks after the treatments and the left testis of remaining lambs were collected after eight weeks and a two-time enzymatic digestion process was used to isolate SSCs. The results showed that all rams that had received intraperitoneal injections of busulfan died. But by testicular injecting of same dose of the drug, 40% of the animals died. The testicular injection of rams with 1, 2 and 4 mg/kg of busulfan resulted in a dose dependent decrease in testis size and also spermatocytes population after 5 weeks of treatments. From the results of colony formation 8 weeks after treatment with busulfan, it can be concluded that only in 1 and 2 mg/kg of busulfan, recovery of endogenous germ cells was performed. In conclusion, the results demonstrated that intra-testicular injections of busulfan (2 mg/kg) reduced spermatocytes population in ram testis within 5 weeks of treatments, and this effect was reversible within 8 weeks of injection. However, it was not recommended to inject 4 mg/kg busulfan into the peritoneal cavity or testis of lambs based on its side effects.

Spermatogonial stem cell survival in ram lambs following busulfan treatment

To clarify the effect of busulfan on the depletion of spermatogonial stem cells (SSCs) from shal rams testis, in the first experiment, lambs were treated by intraperitoneal injection with 4 mg/kg busulfan. In the second experiment, different concentrations of busulfan (1, 2 and 4 mg/kg) were injected directly into both sides of the left testis. The testes of 8 lambs were collected by standard castration procedure for histological analysis five weeks after the treatments and the left testis of remaining lambs were collected after eight weeks and a two-time enzymatic digestion process was used to isolate SSCs. The results showed that all rams that had received intraperitoneal injections of busulfan died. But by testicular injecting of same dose of the drug, 40% of the animals died. The testicular injection of rams with 1, 2 and 4 mg/kg of busulfan resulted in a dose dependent decrease in testis size and also spermatocytes population after 5 weeks of treatments. From the results of colony formation 8 weeks after treatment with busulfan, it can be concluded that only in 1 and 2 mg/kg of busulfan, recovery of endogenous germ cells was performed. In conclusion, the results demonstrated that intra-testicular injections of busulfan (2 mg/kg) reduced spermatocytes population in ram testis within 5 weeks of treatments, and this effect was reversible within 8 weeks of injection. However, it was not recommended to inject 4 mg/kg busulfan into the peritoneal cavity or testis of lambs based on its side effects.(AU)

The Sertoli cell: what can we learn from different vertebrate models?

Besides having medical applications, comparative studies on reproductive biology are very useful, providing, for instance, essential knowledge for basic, conservation and biotechnological research. In order to maintain the reproductive potential and the survival of all vertebrate species, both sperm and steroid production need to occur inside the testis. From the approximately fifty thousand vertebrate species still alive, very few species are already investigated; however, our knowledge regarding Sertoli cell biology is quite good. In this regard, it is already known that since testis differentiation the Sertoli cells are the somatic cells in charge of supporting and orchestrating germ cells during development and full spermatogenesis in adult animals. In the present review, we highlight key aspects related to Sertoli cell biology in vertebrates and show that this key testis somatic cell presents huge and intrinsic plasticity, particularly when cystic (fish and amphibians) and non-cystic (reptiles, birds and mammals) spermatogenesis is compared. In particular, we briefly discuss the main aspects related to Sertoli cells functions, interactions with germ cells, Sertoli cells proliferation and efficiency, as well as those regarding spermatogonial stem cell niche regulation, which are crucial aspects responsible for the magnitude of sperm production. Most importantly, we show that we could greatly benefit from investigations using different vertebrate experimental models, mainly now that there is a big concern regarding the decline in human sperm counts caused by a multitude of factors.

The Sertoli cell: what can we learn from different vertebrate models?

Besides having medical applications, comparative studies on reproductive biology are very useful, providing, for instance, essential knowledge for basic, conservation and biotechnological research. In order to maintain the reproductive potential and the survival of all vertebrate species, both sperm and steroid production need to occur inside the testis. From the approximately fifty thousand vertebrate species still alive, very few species are already investigated; however, our knowledge regarding Sertoli cell biology is quite good. In this regard, it is already known that since testis differentiation the Sertoli cells are the somatic cells in charge of supporting and orchestrating germ cells during development and full spermatogenesis in adult animals. In the present review, we highlight key aspects related to Sertoli cell biology in vertebrates and show that this key testis somatic cell presents huge and intrinsic plasticity, particularly when cystic (fish and amphibians) and non-cystic (reptiles, birds and mammals) spermatogenesis is compared. In particular, we briefly discuss the main aspects related to Sertoli cells functions, interactions with germ cells, Sertoli cells proliferation and efficiency, as well as those regarding spermatogonial stem cell niche regulation, which are crucial aspects responsible for the magnitude of sperm production. Most importantly, we show that we could greatly benefit from investigations using different vertebrate experimental models, mainly now that there is a big concern regarding the decline in human sperm counts caused by a multitude of factors.(AU)

Recent developments in the spermatogonial stem cell field

This review aims at putting in perspective the many new developments in our understanding of spermatogonial multiplication and stem cell renewal in non-primate mammals. In the rodent seminiferous epithelium, the spermatogonial compartment can be subdivided into A, In and B spermatogonia, that show no, some or abundant nuclear het erochromatin, respectively. At first, it was thought that all A spermatogonia were spermatogonial stem cells while In and B spermatogonia were in the differentiation pathway. Then there appeared to be a class of so -called undifferentiated A spermatogonia, subdivided according to their topographical arrangement in to singles (As), pairs (Apr) and chains of 4, 8 and 16 A Al spermatogonia. Four (in mouse and rat) subsequent generations of A spermatogonia together with In and B spermatogonia were called differentiating type spermatogonia. A socalled As model was proposed in which the As spermatogonia are the stem cells that self -renew by forming new singles or give rise to Apr spermatogonia that eventually will become spermatozoa. The As model was challenged by the fragmentation model in which stem cell renewal was supposed to occur by way of fragmentation of clones of A al spe rmatogonia.

Recent developments in the spermatogonial stem cell field

This review aims at putting in perspective the many new developments in our understanding of spermatogonial multiplication and stem cell renewal in non-primate mammals. In the rodent seminiferous epithelium, the spermatogonial compartment can be subdivided into A, In and B spermatogonia, that show no, some or abundant nuclear het erochromatin, respectively. At first, it was thought that all A spermatogonia were spermatogonial stem cells while In and B spermatogonia were in the differentiation pathway. Then there appeared to be a class of so -called undifferentiated A spermatogonia, subdivided according to their topographical arrangement in to singles (As), pairs (Apr) and chains of 4, 8 and 16 A Al spermatogonia. Four (in mouse and rat) subsequent generations of A spermatogonia together with In and B spermatogonia were called differentiating type spermatogonia. A socalled As model was proposed in which the As spermatogonia are the stem cells that self -renew by forming new singles or give rise to Apr spermatogonia that eventually will become spermatozoa. The As model was challenged by the fragmentation model in which stem cell renewal was supposed to occur by way of fragmentation of clones of A al spe rmatogonia.(AU)

Condição metabólica e integridade das membranas, plasmática e acrossomal, da célula espermática bovina após criopreservação em diferentes meios diluidores acrescidos com diferentes tipos de insulina / Metabolic condition and integrity of membranes, and plasma acrosomal, cell bovine sperm after cryopreservation in different extenders greater with different kinds of insulin

Seminal plasma is a fluid with essential role in sperm functions in vivo, from ejaculation to fertilization.Among the hormones present in this medium, insulin stands out, due to its key role in the structure and motility,favoring fertilization. Insulin acts as preservation factor not able sperm and may be the crux of spermpreservation when using cryogenic processes. This study aimed to evaluate the metabolic and structuralcondition of bovine sperm cells after freezing using extenders plus different types of insulin and different types ofegg yolk. Our results showed that sperm motility was not affected by treatments used (insulin and yolk). Inconclusion, the type of egg yolk, the paw (Anas platyrhynchos), increased the viability of cryopreserved spermcells, regardless of the type of insulin used.(AU)

Condição metabólica e integridade das membranas, plasmática e acrossomal, da célula espermática bovina após criopreservação em diferentes meios diluidores acrescidos com diferentes tipos de insulina / Metabolic condition and integrity of membranes, and plasma acrosomal, cell bovine sperm after cryopreservation in different extenders greater with different kinds of insulin

Seminal plasma is a fluid with essential role in sperm functions in vivo, from ejaculation to fertilization.Among the hormones present in this medium, insulin stands out, due to its key role in the structure and motility,favoring fertilization. Insulin acts as preservation factor not able sperm and may be the crux of spermpreservation when using cryogenic processes. This study aimed to evaluate the metabolic and structuralcondition of bovine sperm cells after freezing using extenders plus different types of insulin and different types ofegg yolk. Our results showed that sperm motility was not affected by treatments used (insulin and yolk). Inconclusion, the type of egg yolk, the paw (Anas platyrhynchos), increased the viability of cryopreserved spermcells, regardless of the type of insulin used.

Regulation of germ line stem cell homeostasis

Mammalian spermatogenesis is a complex process in which spermatogonial stem cells of the testis (SSCs) develop to ultimately form spermatozoa. In the seminiferous epithelium, SSCs self-renew to maintain the pool of stem cells throughout life, or they differentiate to generate a large number of germ cells. A balance between SSC self-renewal and differentiation is therefore essential to maintain normal spermatogenesis and fertility. Stem cell homeostasis is tightly regulated by signals from the surrounding microenvironment, or SSC niche. By physically supporting the SSCs and providing them with these extrinsic molecules, the Sertoli cell is the main component of the niche. Earlier studies have demonstrated that GDNF and CYP26B1, produced by Sertoli cells, are crucial for self-renewal of the SSC pool and maintenance of the undifferentiated state. Down-regulating the production of these molecules is therefore equally important to allow germ cell differentiation. We propose that NOTCH signaling in Sertoli cells is a crucial regulator of germ cell fate by counteracting these stimulatory factors to maintain stem cell homeostasis. Dysregulation of this essential niche component can lead by itself to sterility or facilitate testicular cancer development.

Regulation of germ line stem cell homeostasis

Mammalian spermatogenesis is a complex process in which spermatogonial stem cells of the testis (SSCs) develop to ultimately form spermatozoa. In the seminiferous epithelium, SSCs self-renew to maintain the pool of stem cells throughout life, or they differentiate to generate a large number of germ cells. A balance between SSC self-renewal and differentiation is therefore essential to maintain normal spermatogenesis and fertility. Stem cell homeostasis is tightly regulated by signals from the surrounding microenvironment, or SSC niche. By physically supporting the SSCs and providing them with these extrinsic molecules, the Sertoli cell is the main component of the niche. Earlier studies have demonstrated that GDNF and CYP26B1, produced by Sertoli cells, are crucial for self-renewal of the SSC pool and maintenance of the undifferentiated state. Down-regulating the production of these molecules is therefore equally important to allow germ cell differentiation. We propose that NOTCH signaling in Sertoli cells is a crucial regulator of germ cell fate by counteracting these stimulatory factors to maintain stem cell homeostasis. Dysregulation of this essential niche component can lead by itself to sterility or facilitate testicular cancer development.(AU)

Endocrine and paracrine regulation of zebrafish spermatogenesis: the Sertoli cell perspective

Spermatogonial stem cells (SSCs) either self-renew or differentiate into spermatogonia that further develop into spermatozoa. Self-renewal occurs when residing in a specific micro-environment (niche) While displacement from the niche would tip the signalling balance towards differentiation. Considering the cystic type of spermatogenesis in fish, the SSC candidates are single type A undifferentiated (Aund) spermatogonia, enveloped by mostly one niche-forming Sertoli cell. When going through a self-renewal cell cycle, the resulting new single type Aund spermatogonium would have to recruit another Sertoli cell to expand the niche space, while a differentiating germ cell cyle would result in a pair of spermatogonia that remain in contact with their cyst-forming Sertoli cells. In zebrafish, thyroid hormone stimulates the proliferation of Sertoli cells and of type Aund spermatogonia, involving Igf3, a new member of the Igf family. In cystic spermatogenesis, type A und spermatogonia usually do not leave the niche, so that supposedly the signalling in the niche changes when switching from self-renewal to differentiation. Recombinant zebrafish (rz) Fsh down-regulated Sertoli cell anti-müllerian hormone (amh) mRNA levels, and rzAmh inhibited differentiation of type A und spermatogonia as well as Fsh-stimulated steroidogenesis. Thus, for Fsh to efficiently stimulate testis functions, Amh bioactivity should be dampened. We also discovered that Fsh increased Sertoli cell Igf3 gene and protein expression; rzIgf3 stimulated spermatogonial proliferation and Fsh-stimulated spermatogenesis was significantly impaired by inhibiting Igf receptor signaling. We propose that in zebrafish, Fsh is the major regulator of testis functions and, supported by other endocrine systems (e.g. thyroid hormone), regulates Leydig cell steroidogenesis as well as Sertoli cell number and growth factor production to promote spermatogenesis.

Endocrine and paracrine regulation of zebrafish spermatogenesis: the Sertoli cell perspective

Spermatogonial stem cells (SSCs) either self-renew or differentiate into spermatogonia that further develop into spermatozoa. Self-renewal occurs when residing in a specific micro-environment (niche) While displacement from the niche would tip the signalling balance towards differentiation. Considering the cystic type of spermatogenesis in fish, the SSC candidates are single type A undifferentiated (Aund) spermatogonia, enveloped by mostly one niche-forming Sertoli cell. When going through a self-renewal cell cycle, the resulting new single type Aund spermatogonium would have to recruit another Sertoli cell to expand the niche space, while a differentiating germ cell cyle would result in a pair of spermatogonia that remain in contact with their cyst-forming Sertoli cells. In zebrafish, thyroid hormone stimulates the proliferation of Sertoli cells and of type Aund spermatogonia, involving Igf3, a new member of the Igf family. In cystic spermatogenesis, type A und spermatogonia usually do not leave the niche, so that supposedly the signalling in the niche changes when switching from self-renewal to differentiation. Recombinant zebrafish (rz) Fsh down-regulated Sertoli cell anti-müllerian hormone (amh) mRNA levels, and rzAmh inhibited differentiation of type A und spermatogonia as well as Fsh-stimulated steroidogenesis. Thus, for Fsh to efficiently stimulate testis functions, Amh bioactivity should be dampened. We also discovered that Fsh increased Sertoli cell Igf3 gene and protein expression; rzIgf3 stimulated spermatogonial proliferation and Fsh-stimulated spermatogenesis was significantly impaired by inhibiting Igf receptor signaling. We propose that in zebrafish, Fsh is the major regulator of testis functions and, supported by other endocrine systems (e.g. thyroid hormone), regulates Leydig cell steroidogenesis as well as Sertoli cell number and growth factor production to promote spermatogenesis.(AU)

An overview of spermatogonial stem cell physiology, niche and transplantation in fish

Similar to mammals, spermatogenesis in fish is initiated by spermatogonial stem cells (SSCs) which either self-renew or gradually differentiate to produce mature sperm. SSCs are located in a particular testis microenvironment called SSC ni che, formed by Sertoli and peritubular myoid cells, the basement membrane and other cellular components/factors from the intertubular compartment that regulate SSCs maintenance and fate. Considering the great variation in testis structure/arrangemen t across fish species, the study of the niche components is crucial to understand SSCs physiology. Additionally, the germ cell transplantation technique, which has been applied to fish in the last decade, is a unique approach to elucidating important functional aspects of SSCs biology such as: (i) the capacity of SSCs to colonize the testis of recipient species (syngeneic and xenogeneic transplantation) giving rise to donor sperm; (ii) the plasticity of these cells, considering that spermatogonia and oogonia can be derived from SSCs collected from the opposite sex; and (iii) the possibility of genetically manipulating SSCs before transplantation to produce transgenic fish. However, fish SSC isolation and characterization has been lim ited so far by the lack of specific molecular markers fo r these cells. Therefore, various research groups are currently investigating specific SSCs markers and, up to date, few proteins have been identified in different spermatogonial populations from distinct fish species (e.g. Notch1, Ly75, Plzf, Oct-4, SGSA -1). Furthermore, the development of a fish SSC culture system would allow the investigation of important regulatory aspects of the SSC physiology in well-defined conditions as well as to in vitro amplify these rare cells. Overall, the study of SSC physiology, niche and transplantation in fish has opened up new scenarios for the development of aquaculture and reproductive biotechnologies such as germplasm conservation of endangered or commercially important species and the possibility of generating transgenic fish.

An overview of spermatogonial stem cell physiology, niche and transplantation in fish

Similar to mammals, spermatogenesis in fish is initiated by spermatogonial stem cells (SSCs) which either self-renew or gradually differentiate to produce mature sperm. SSCs are located in a particular testis microenvironment called SSC ni che, formed by Sertoli and peritubular myoid cells, the basement membrane and other cellular components/factors from the intertubular compartment that regulate SSCs maintenance and fate. Considering the great variation in testis structure/arrangemen t across fish species, the study of the niche components is crucial to understand SSCs physiology. Additionally, the germ cell transplantation technique, which has been applied to fish in the last decade, is a unique approach to elucidating important functional aspects of SSCs biology such as: (i) the capacity of SSCs to colonize the testis of recipient species (syngeneic and xenogeneic transplantation) giving rise to donor sperm; (ii) the plasticity of these cells, considering that spermatogonia and oogonia can be derived from SSCs collected from the opposite sex; and (iii) the possibility of genetically manipulating SSCs before transplantation to produce transgenic fish. However, fish SSC isolation and characterization has been lim ited so far by the lack of specific molecular markers fo r these cells. Therefore, various research groups are currently investigating specific SSCs markers and, up to date, few proteins have been identified in different spermatogonial populations from distinct fish species (e.g. Notch1, Ly75, Plzf, Oct-4, SGSA -1). Furthermore, the development of a fish SSC culture system would allow the investigation of important regulatory aspects of the SSC physiology in well-defined conditions as well as to in vitro amplify these rare cells. Overall, the study of SSC physiology, niche and transplantation in fish has opened up new scenarios for the development of aquaculture and reproductive biotechnologies such as germplasm conservation of endangered or commercially important species and the possibility of generating transgenic fish.(AU)

Preservação da linhagem germinativa em machos murinos

Ao longo da vida reprodutiva dos machos adultos, espermatozoides são formados pelas células-tronco espermatogoniais (SSCs, do inglês spermatogonial stem cells) por um processo conhecido como espermatogênese. O cultivo in vitro de SSCs abriu novas possibilidades para a preservação da linhagem germinativa, porém os protocolos requerem a adição de fatores de crescimento, o que encarece a manutenção dessas células por um tempo prolongado, fazendo da criopreservação de SSCs uma alternativa para esse problema. A literatura científica ainda não definiu uma metodologia que seja eficaz na congelação das SSCs, bem como na seleção de uma população pura, ou seja, sem contaminação por células que possam causar carcinomas. O presente estudo teve por objetivo avaliar duas metodologias de vitrificação de tecido testicular de murinos. Para testar esse objetivo, testículos de camundongos da linhagem C57BL6 GFP+ com 8 a 10 dias de idade foram submetidos a dois protocolos (Protocolo 1 e Protocolo 2) de vitrificação de tecido testicular descritos na literatura. Após 4 a 12 semanas, os testículos vitrificados foram aquecidos, reidratados e as células testiculares dissociadas para avaliação da viabilidade e concentração. As SSCs foram selecionadas por meio de separação celular por beads magnéticas (MACS) com anticorpo Thy1.2 e transplantadas para testículos de camundongos adultos da linhagem C57BL6 previamente tratados com o quimioterápico busulfan. Após seis semanas, os testículos destes animais foram coletados e os túbulos seminíferos dissociados para avaliação da formação de colônias pelas SSCs transplantadas. Houve diferença estatística (p<0,0001) na viabilidade das células entre o Protocolo 1 e Protocolo 2, sendo de 74,4% e 82,8%, respectivamente. Também houve diferença estatística (p<0,0001) na concentração de células obtidas entre o Protocolo 1 e Protocolo 2, sendo de 0,43x106 e 1,35x106, respectivamente. Colônias formadas pelas SSCs transplantadas foram encontradas nos testículos dos animais transplantadas para os dois protocolos de vitrificação. De forma descritiva, podemos relatar que o número de colônias observadas para as células do Protocolo 2 foi maior comparado ao Protocolo 1. Portanto, conclui-se que os protocolos de vitrificação de tecido testicular de camundongos estudados foram capazes de preservar a viabilidade de células-tronco espermatogoniais possibilitando a formação de colônias por estas, sendo que o Protocolo 2 de vitrificação foi mais eficiente.

Throughout the reproductive life of adult males, spermatozoa are formed by spermatogonial stem cells (SSCs) by a process known as spermatogenesis. The in vitro culture of SSCs created new possibilities for preservation of the germ line, but the protocols require the addition of growth factors, which increases the maintenance costs of these cells for a prolonged time, making of the SSCs cryopreservation an alternative for this problem. The scientific literature has not yet defined a methodology that is effective in the freezing of SSCs, as well as in the selection of a pure population, that is, without contamination by cells that involves the carcinomas. The present study aimed to evaluate two methodologies of vitrification of murine testicular tissue. To test this aim, testes of mice of the C57BL6 GFP + strain with 8 to 10 days old were submitted to two protocols (Protocol 1 and Protocol 2) of testicular tissue vitrification. After 4 to 12 weeks, the vitrified testes were warmed, rehydrated and the testicular cells dissociated for viability and concentration assessment. The SSCs were selected by magnetic-activated cell sorting (MACS) using Thy1.2 antibody and transplanted to testes of adult mice of the C57BL6 strain previously treated with the quimioterapic busulfan. After six weeks, the testes of these animals were collected and seminiferous tubules dissociated to evaluate the formation of colonies by transplanted SSCs. There was a statistical difference (p<0.0001) in cell viability between Protocol 1 and Protocol 2, being 74.4% and 82.8%, respectively. There was also a statistical difference (p<0.0001) in the concentration of cells obtained between Protocol 1 and Protocol 2, being 0.43x106 and 1.35x106, respectively. Colonies formed by the transplanted SSCs were found in the testes of the transplanted animals for the two vitrification protocols. In a descriptive way, we can report that the number of colonies observed for Protocol 2 cells was higher compared to the Protocol 1. Therefore, it is concluded that the mice testicular tissue vitrification protocols studied were able to preserve the viability of spermatogonial stem cells allowing the formation of colonies by these cells and the vitrification Protocol 2 was more efficient.