Isolation and Short-Term Culture of Mouse Spermatocytes for Analysis of Meiosis

Abstract

Understanding meiosis is facilitated by in vitro experimental approaches, but this has not been easily applicable to mammalian meiocytes. Available methods for in vitro analysis of mammalian oocytes are generally limited to experimental analysis of the late prophase period. Short-term cultures of male germ cells have been useful for analysis of earlier meiotic prophase pathways, as well as onset of the meiotic division phase, but no studies have achieved reliable spermatogenesis in vitro. Here we describe a method for preparing highly enriched pachytene spermatocytes from mouse testicular cell suspensions using cell-size fractionation by sedimentation through a bovine serum albumin gradient at unit gravity. We also provide a procedure for short-term culture of spermatocytes and the pharmacological induction of the prophase-to-division phase transition.

In vitro production of fertile sperm from murine spermatogonial stem cell lines

“…Until day 3, most GS cells resided on the basement membrane. They also appeared to have started proliferation at around day 7 some seminiferous tubules were occupied by GS cells (Fig. 1c).…”

“…These data strongly suggest that in vitro meiosis from GS cells took place in the explanted testis tissues. To confirm that the meiosis was genuine, we employed an immunohistochemical technique using an antibody for synaptonemal complex protein 1 (SYCP1) 7 , a specific marker of meiosis, and another for GFP. The SYCP1-antibody demonstrated a spread of chromosomes typical of meiosis in spermatocytes, while the GFPantibody revealed an acrosomal dot in spermatids when Acr-GS cells were incubated in the explants ( Fig.…”

“…Trials to achieve spermatogenesis in vitro, have faced many difficulties and remained incomplete 6,7 . On the other hand, the in vitro proliferation of murine SSCs has become possible by adding glial cell line-derived neurotrophic factor to the SSCs culture media 8,9 .…”

Heat Shock Protein 90 Functions to Stabilize and Activate the Testis-specific Serine/Threonine Kinases, a Family of Kinases Essential for Male Fertility

“…Male germ cells were isolated by enzymatic dissociation of testes from 8 -12-week-old mice, and the cells were separated by sedimentation at unit gravity in 2-4% BSA gradient in a STA-PUT apparatus (ProScience Inc., GlassShop, Toronto, ON, Canada) according to the method described by La Salle et al (39). After sedimentation, the fractions were examined under a microscope, and the germ cell types were identified based on size and the morphological criteria as described by Bellvé (40).…”

Connecting cis-elements and trans-factors with mechanisms of developmental regulation of mRNA translation in meiotic and haploid mammalian spermatogenic cells

“…Similarly, populations of singlecell pachytene spermatocytes can be cultured in 2D culture for 24-36 h and induced with okadaic acid to progress to an abnormal meiotic metaphase I (La Salle et al 2009, Paronetto et al 2009). These culture systems might be useful in studies of the factors and elements that promote the completion of meiosis and round spermatid stage.…”