simple-efficient-technique-for-preparation-testicular-cell

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

Instituto de Investigaciones Biológicas Clemente Estable (IIBCE)

Universidad de la República

Using mRNA differential display and cDNA library screening approaches we have identified differential gene expression of pecanex 1--a mammalian homologue of pecanex gene from Drosophila--in the testes of the rat. Northern blot analyses showed that the transcript is only present in the germ line and not in the somatic cells of the testis, reaching its peak at the pachytene stage of the meiotic prophase. Moreover, nonradioactive in situ hybridization did not detect the expression of the gene in any cell type of the testis other than pachytene spermatocytes. Northern blot assays did not allow the detection of the transcript in nine other tissues. Remarkably, although pecanex exerts a neurogenic role in Drosophila, the transcript was not detectable by Northern blotting in the nervous tissue of adult rats, nor in the brain of neonate and embryonal stages. The protein product of the pecanex 1 gene was detected by immunoblotting in pachytene spermatocytes and round spermatids as well, but not in liver nor brain. From genomic analysis we conclude that, although only one pecanex gene exists in Drosophila, mammalian pecanex 1 belongs to a gene family with three related genes in different chromosomes. We speculate that pecanex 1 could play an important role in the testis, related to spermatogenesis.

The mammalian gene pecanex 1 is differentially expressed during spermatogenesis.

Homogeneity of cell populations is a prerequisite for the analysis of biochemical and molecular events during male gamete differentiation. Given the complex organization of the mammalian testicular tissue, various methods have been used to obtain enriched or purified cell populations, including flow cell sorting. Current protocols are usually time-consuming and may imply loss of short-lived RNAs, which is undesirable for expression profiling. We describe an optimized method to speed up the preparation of suitable testicular cell suspensions for cytometric analysis of different spermatogenic stages from rodents. The procedure takes only 15Â min including testis dissection, tissue cutting, and processing through the Medimachine System (Becton Dickinson). This method could be a substitute for the more tedious and time-consuming cell preparation techniques currently in use.

Ultra-fast and optimized method for the preparation of rodent testicular cells for flow cytometric analysis.

Spats1 encodes the first reported testis-specific protein containing a long serine stretch. Besides, it bears a probable bipartite nuclear localization signal. Here, we describe the expression pattern of Spats1 in rat along embryonic and postnatal testis development by immunoblots and confocal immunohistochemistry. Spats1 is first expressed in the embryo at 17.5 days post-coitum, coinciding with the time when gonocytes acquire a quiescent state. At this time expression is detected in peritubular myoid cells and gonocytes. Spats1 attains maximum levels during meiosis of the first spermatogenic wave, mainly in pachytene spermatocytes, while a lower signal is also observed in spermatogonia, Sertoli cells and myoid cells. Protein levels dramatically decay afterwards, with minimum expression in adult individuals, where no signal was detected in elongating spermatids or spermatozoa. Spats1 is mostly cytoplasmic, although in pachytene spermatocytes it mapped to nuclei as well. Alkaline phosphatase treatment showed that this protein would be highly phosphorylated. Moreover, we show that the protein is highly conserved along metazoan evolution. Our results suggest a role in the initiation of the first spermatogenic wave, and in the establishment or progression of the first male meiotic division.

Spats 1 (Srsp1) is differentially expressed during testis development of the rat.

Mammalian spermatogenesis is still nowadays poorly understood at the molecular level. Testis cellular heterogeneity is a major drawback for spermatogenic gene expression studies, especially when research is focused on stages that are usually very short and poorly represented at the cellular level such as initial meiotic prophase I (i.e., leptotene [L] and zygotene [Z]). Presumably, genes whose products are involved in critical meiotic events such as alignment, pairing and recombination of homologous chromosomes are expressed during the short stages of early meiotic prophase. Aiming to characterize mammalian early meiotic gene expression, we have found the guinea pig (Cavia porcellus) as an especially attractive model. A detailed analysis of its first spermatogenic wave by flow cytometry (FCM) and optical microscopy showed that guinea pig testes exhibit a higher representation of early meiotic stages compared to other studied rodents, partly because of their longer span, and also as a result of the increased number of cells entering meiosis. Moreover, we have found that adult guinea pig testes exhibit a peculiar 4C DNA content profile, with a bimodal peak for L/Z and P spermatocytes that is absent in other rodents. Besides, we show that this unusual 4C peak allows the separation by FCM of highly pure L/Z spermatocyte populations aside from pachytene ones, even from adult individuals. To our knowledge, this is the first report on an accurate and suitable method for highly pure early meiotic prophase cell isolation from adult mammals, and thus sets an interesting approach for gene expression studies aiming at a deeper understanding of the molecular groundwork underlying male gamete production.

High-purity flow sorting of early meiocytes based on DNA analysis of guinea pig spermatogenic cells.

We report the development of a 3-week laboratory activity for an undergraduate molecular biology course. This activity introduces students to the practice of basic molecular techniques such as restriction enzyme digestion, agarose gel electrophoresis, cloning, plasmid DNA purification, Southern blotting, and sequencing. Students learn how to carry out a GenBankâ„¢ search as they are encouraged to compile most available information about their cloned sequence. The natural integration of the bench work with the use of the bioinformatics tools is considered a major advantage of this laboratory course.

Article	Year
The mammalian gene pecanex 1 is differentially expressed during spermatogenesis. Biochimica et biophysica acta\| PubMed ID: 15777640	2005
Ultra-fast and optimized method for the preparation of rodent testicular cells for flow cytometric analysis. Biological procedures online\| PubMed ID: 19495915	2009
Spats 1 (Srsp1) is differentially expressed during testis development of the rat. Gene expression patterns : GEP\| PubMed ID: 19948251	2010
High-purity flow sorting of early meiocytes based on DNA analysis of guinea pig spermatogenic cells. Cytometry. Part A : the journal of the International Society for Analytical Cytology\| PubMed ID: 21520399	2011
Molecular cloning and analysis of a DNA repetitive element from the mouse genome. Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology\| PubMed ID: 21638677	2006

Adriana Geisinger

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