In vitro mouse spermatogenesis with an organ culture method in chemically defined medium

Abstract

We previously reported the successful induction and completion of mouse spermatogenesis by culturing neonatal testis tissues. The culture medium consisted of α-minimum essential medium (α-MEM), supplemented with Knockout serum replacement (KSR) or AlbuMAX, neither of which were defined chemically. In this study, we formulated a chemically defined medium (CDM) that can induce mouse spermatogenesis under organ culture conditions. It was found that bovine serum albumin (BSA) purified through three different procedures had different effects on spermatogenesis. We also confirmed that retinoic acid (RA) played crucial roles in the onset of spermatogonial differentiation and meiotic initiation. The added lipids exhibited weak promoting effects on spermatogenesis. Lastly, luteinizing hormone (LH), follicle stimulating hormone (FSH), triiodothyronine (T3), and testosterone (T) combined together promoted spermatogenesis until round spermatid production. The CDM, however, was not able to produce elongated spermatids. It was also unable to induce spermatogenesis from the very early neonatal period, before 2 days postpartum, leaving certain factors necessary for spermatogenic induction in mice unidentified. Nonetheless, the present study provided important basic information on testis organ culture and spermatogenesis in vitro.

In vitro spermatogenesis in two‐dimensionally spread mouse testis tissues

“…It was classified into 6 grades, 0‐5, based on the expression area: 0, ~20, ~40, ~60, ~80, and‐100%, respectively. This GFP grading scale faithfully corresponded to spermatogenic progression, as confirmed in previous studies . To identify haploid cells with an acrosome cap structure, cultured tissues were observed with an inverted microscope applying the GFP‐excitation light (Olympus IX 73; Olympus).…”

“…As for evaluation, GFP expression extent overall in each tissue was used. Specifically, the cultured tissues were recorded as photograph every week and the proportion of GFP‐expressing region in each tissue was estimated and classified into 6 grades: 0, ~20, ~40, ~60, ~80, and ~100% . Contrary to the control of AG, whose central area usually lacked GFP expression, samples in the PC groups showed GFP expression rather uniformly throughout the whole tissue in many cases, although the central area is yet weaker in GFP (Figure A).…”

“…Testes were decapsulated and the tissue was divided into several pieces of appropriate size with forceps. Tissues were placed on the agarose gel block (1.5% w/v) half‐soaked in the culture medium in each well of a 12‐well plate (CELLSTAR® Tissue Culture Plates; Greiner Bio‐One) . They were incubated as they were, AG group, or covered with PC chip, PC group.…”

In vitro spermatogenesis: A century‐long research journey, still half way around

“…Because KSR was supposed to contain AlbuMAX, the reason of the success was attributable to AlbuMAX. We have further showed that AlbuMAX, but not other popular bovine serum albumin (BSA), has such a strong effect to induce and maintain spermatogenesis . In addition, another BSA product, purified by chromatographic method like the preparation of AlbuMAX, also has a similar effect.…”

“…We have further showed that AlbuMAX, but not other popular bovine serum albumin (BSA), has such a strong effect to induce and maintain spermatogenesis. 33 In addition, another BSA product, purified by chromatographic method like the preparation of AlbuMAX, also has a similar effect. Thus, we speculate that chromatography purified BSAs contain some important factors effective for in vitro spermatogenesis.…”