Mariana Ianello Giassetti

Effect of age on expression of spermatogonial markers in bovine testis and isolated cells

Spermatogonial stem cells (SSC) are the most undifferentiated germ cell present in adult male testes and, it is responsible to maintain the spermatogenesis. Age has a negative effect over stem cell, but the aging effect on SSC is not elucidated for bovine. The present study aim to evaluate the effect of age on the expression of undifferentiated spermatogonial markers in testis and in enriched testicular cells from prepubertal calves and adult bulls. In this matter, testicular parenchyma from calves (3-5 months) (n=5) and bulls with 3 years of age (n=5) were minced and, isolated cells were obtained after two enzymatic digestions. Differential platting was performed for two hours onto BSA coated dish. Cell viability was assessed by Trypan Blue solution exclusion method and testicular cells enriched for SSC was evaluated by expression of specific molecular markers by qRT-PCR (POU5F1, GDNF, CXCR4, UCHL1, ST3GAL, SELP, ICAM1 and ITGA6) and flow cytometry (GFRA1, CXCR4 and ITGA6). CXCR4 and UCHL1 expression was evaluated in fixated testes by immunohistochemistry. We observed that age just affected the expression of selective genes [SELP (Fold Change=5.61; p=0.0023) and UCHL1 (Fold Change=4.98; p=0.0127)]. By flow cytometry, age affected only the proportion of ITGA6+ cells (P<0.001), which was higher in prepubertal calves when compared to adult bulls. In situ, we observed an effect of age on the number of UCHL1+ (p=0.0006) and CXCR4+ (p=0.0139) cells per seminiferous tubule. At conclusion, age affects gene expression and the population of cells expressing specific spermatogonial markers in the bovine testis.

Spermatogonial Stem Cells and Animal Transgenesis

Spermatogonial stem cells (SSCs) are unipotent adult stem cells responsible for the maintenance of the spermatogenesis throughout the entire life of the male. We could say that the mammalian spermatogenesis is a classic adult stem cell-dependent process, sustained by self renewal and differentiation of SSCs. They are the only germline stem cells in adults. These cells can be found in the seminiferous tubule, lying near to the basement membrane. The SSC may choose to self-renewal or generate a daughter cell committed to differentiation. Studying SSCs provides a model to better understand adult stem cell biology and decipher the mechanisms that control SSC functions. It was reported that these cells hold the ability to colonize the seminiferous tubules after transplantation, restoring spermatogenesis. Besides the biomedical potential to perform studies of infertility in many species, SSCs present a promising application in biotechnology in the production of transgenic animals. This alternative route for transgenesis is of interest because a single male will generate by regular mate a variety of transgenic progenies. The production of a transgenic gonad can overcome the obstacles faced with the sperm-mediated gene transfer (SMGT) due to the high specialization of sperm. The use of SSC for transgenesis relies on targeting a much more undifferentiated germ cell and the potential permanent modification of the germ line. In this manner, this chapter aims to review the following topics regarding SSCs: (1) Mammalian spermatogenesis and SSCs; (2) Characterization of SSCs; (3) Isolation and in vitro culture of SSCs; (4) Transplantation of SSCs and animal transgenesis.

Comparison of Diverse Differential Plating Methods to Enrich Bovine Spermatogonial Cells.

Spermatogonial stem cells (SSC) have important applications in domestic animal reproduction and advanced biotechnologies. Because differential plating is one of the most common methods used for SSC enrichment, the goal of this study was to compare three differential plating methods for the enrichment of bovine SSC. To achieve this goal, testicular parenchyma from pre-pubertal calves was minced and single cells were obtained after two enzymatic digestions. We compared three coating methods for differential plating: laminin (20 ng/ml), BSA (0.05 mg/ml) and PBS. Cells were incubated at 37°C, 5% CO2 in air for 15 min onto laminin-coated dishes or 2 h onto BSA- or PBS-coated dishes. Cell viability was assessed by trypan blue exclusion method. Recovered cells were analysed for the expression of SSC molecular markers by quantitative RT-PCR (GFRA1, CXCR4, ITGA6, THY1) and flow cytometry (GFRA1, CXCR4 and ITGA6). Cells at time 0, adherent cells on laminin and non-adherent cells from BSA and PBS groups had the same cell viability (p = 0.0655). GFRA1, CXCR4 and THY1 relative gene expression was higher (p = 0.0402, p = 0.0007, p = 0.0117, respectively) for non-adherent cells selected in PBS group. Flow cytometry analysis revealed that the presence of GFRA-positive (GFRA+) cells was higher in non-adherent cells from BSA and PBS groups (p < 0.001). However, laminin-adherent cells had higher number of ITGA6+ cells (p < 0.001) and lower presence of CXCR4+ cells (p = 0.0012). In conclusion, differential plating is an effective method for the enrichment of bovine undifferentiated spermatogonia and higher expression of SSC markers is obtained without laminin or BSA coating.

Quantificação do interferon-tau durante o reconhecimento materno da gestação em fêmeas bovinas Bos taurus indicus

Durante o periodo critico do reconhecimento materno, compreendido entre o 15o e 19o dias da gestacao, o concepto deve sintetizar competentemente moleculas capazes de bloquear a sintese de prostaglandina F2α (PGF2α) e a luteolise. Em bovinos, a principal macromolecula proteica envolvida em tal bloqueio e o interferon-tau (IFN-τ). Durante o periodo critico, falhas neste reconhecimento determinam a mortalidade embrionaria em ate 40% das femeas inseminadas. Informacoes sobre o IFN-τ em animais Bos taurus indicus, ainda sao restritas. Este estudo objetivou uma avaliacao quantitativa do IFN-τ durante o periodo critico do reconhecimento materno, em lavados uterinos obtidos por sonda de Foley (dias 14, 16 e 18 posestro) ou post-mortem (dia 18 pos-estro). Para tanto, foram utilizadas femeas multiparas azebuadas (Bos taurus indicus), ciclicas ou prenhes, nos dias 14, 16 e 18 pos-estro. Para a obtencao dos lavados, os uteros foram infundidos com solucao de Ringer Simples. Os lavados foram concentrados por ultra-filtracao e liofilizados. As macromoleculas proteicas foram separadas por Eletroforese Unidimensional SDSPAGE, em gel com 15% de poliacrilamida. A quantificacao do IFN-τ nos lavados uterinos foi realizada por Western-Blotting e densitometria. Tanto nos lavados obtidos por sonda de Foley quanto nos post-mortem foi possivel observar bandas de proteinas que apresentaram reacao cruzada com os anticorpos utilizados no Western-Blotting. O IFN-τ foi detectado apenas nos lavados uterinos post-mortem de vacas prenhes (P<0,05). A densidade optica nao foi afetada pelo dia do periodo critico, estado (ciclico ou prenhe) ou interacao dia x estado. Nos lavados post-mortem nao houve efeito de peso do concepto ou concentracao de progesterona plasmatica no dia do lavado na densidade da banda proteica referente ao IFN-τ . Concluiu-se que a deteccao e quantificacao do IFN-τ no ambiente uterino de vacas azebuadas, nestas condicoes experimentais, e possivel apenas em lavados uterinos obtidos post-mortem.

Spermatogonial stem cell potential of CXCR4-positive cells from prepubertal bull testes

Spermatogonial stem cells (SSC) have the potential to restore spermatogenesis when transplanted into testes depleted of germ cells. Due to this property, SSC could be used in breeding programs and in transgenic animal research. Particularly in cattle, SSC are not as well characterized as in mice or humans. In mice, C-X-C Motif Chemokine Receptor 4 positive (CXCR4+) testicular cells have high SSC potential. It, therefore, was hypothesized that CXCR4 is a marker of undifferentiated spermatogonia in cattle. Using samples from pre-pubertal calves, the CXCR4 protein was detected by immunohistochemistry in a few cells of the seminiferous tubules. Testicular cells were isolated, frozen-thawed and submitted to magnetic-activated cell sorting using anti-CXCR4 antibody. Quantitative RT-PCR analysis revealed that CXCR4+ cells had THY1, OCT4 and ZBTB16 (or PLZF) mRNA in these cells. Flow cytometry results indicated that the proportion of THY1+ cells is enriched in CXCR4+ populations. Colonization potential of CXCR4+ cells was assessed after xenotransplantation into testes of nude mice treated with busulfan. Transplantation of CXCR4+ cells yielded an increase of 5.4-fold when compared to CXCR4- cells. These results indicate that CXCR4 could be used as a marker to enrich and sort cells of bulls with putative spermatogonial stem cell potential.

Genetic Engineering and Cloning: Focus on Animal Biotechnology

Over the last 35 years the term genetic engineering has been commonly used not only in science but also in others parts of society. Nowadays this name is often associated by the media forensic techniques to solve crimes, paternity, medical diagnosis and, gene mapping and sequencing. The popularization of genetic engineering is consequence of its wide use in laboratories around the world and, developing of modern and efficient techniques. The genetic engineering, often used with trivia, involves sophisticated techniques of gene manipulation, cloning and modification. Many authors consider this term as synonymous as genetic modification, where a synthetic gene or foreign DNA is inserted into an organism of interest. Organism that receives this recombinant DNA is considered as genetically modified (GMO). Its production are summarized in simplified form in five steps: 1) Isolation of interested gene, 2) Construction, gene of interested is joined with promoters (location and control the level of expression), terminator (indicates end of the gene) and expression marker (identify the gene expression), 3) transformation (when the recombinant DNA is inserted into the host organism), 4) Selection (selection of those organisms that express the markers), 5) Insertion verification of recombinant DNA and its expression [1].