Expression analysis of PAWP during mouse embryonic stem cell-based spermatogenesis in vitro

Abstract

Postacrosomal sheath WW domain-binding protein (PAWP) is a novel sperm protein identified as a candidate sperm-borne, oocyte-activating factor (SOAF). However, regulation of PAWP gene expression is poorly understood. Therefore, we examined the PAWP gene expression across different stages of mouse embryonic stem cell (ESC)-based spermatogenesis in vitro and compared this expression at different stages of mouse testis development in vivo. Expression of PAWP was also examined in mouse embryonic fibroblasts (MEF), Sertoli cell, and the NIH3T3 cancerous cell line. We used a transgenic mouse ESC line C57BL/6J expressing Stra8-EGFP that was plated in murine ESC medium. To induce differentiation, cells were cultured on gelatin-coated medium with Retinoic Acid (RA) treatment. We applied reverse transcription-PCR and real-time PCR to analyze the differential expression of PAWP mRNA during different stages of mouse ESC differentiation in vitro parallel with mouse testis development in vivo and in cell lines. We found that expression of PAWP is increased during testis development in vivo with greatest expression at postmeiotic phase. It is also highly expressed in mouse ESC-derived germ-like cells after 30 d of RA induction in vitro. PAWP is remarkably expressed in mouse ESC and NIH3T3 cell line. These results indicate that PAWP plays a role in spermatogenesis and germ cell development. Moreover, we suggest PAWP as one of the markers that could be looked in ESC studies as a confirmed testis-specific gene. We also suggest an additional possible role for PAWP in proliferation of cancerous cell in general.

The Evaluation of WBP2NL-Related Genes Expression in Breast Cancer

“…We have previously evaluated the expression of WBP2NL gene in cancerous cell line and breast tumor tissues and reported changes in expression that could contribute to increase cancerous cell proliferation [23], and breast cancer tumorigenesis (Unpublished data). In the present study, we evaluated a wide range of genes expression related to WBP2NL in both breast cancers and normal breasts.…”

“…We have also evaluated the WBP2NL gene expression in cancerous cell line and breast tumor tissues and revealed changes in expression that could contribute to increase cancerous cell proliferation [23], and breast cancer tumorigenesis (Unpublished data). However, the mechanisms of WBP2NL in cancerous cell proliferation and its clinical relevance have not been investigated.…”

Insights into in vitro spermatogenesis in mammals: Past, present, future

“…Retinoic acid‐synthesizing enzymes are expressed in Sertoli cells (Vernet et al, ), suggesting that retinoic acid produced by these somatic cells act on spermatogonia via nuclear receptors to initiate meiosis in the postnatal testis (Akmal, Dufour, & Kim, ; Bowles and Koopman, ; Chen et al, ; Eskild, Ree, Levy, Jahnsen, & Hansson, ). Based on these documented effects by retinoic acid, our group and others use this molecule to induce meiosis during in vitro spermatogenesis (Nayernia, Nolte, ; Nourashrafeddin, Aarabi, ; Nourashrafeddin, Ebrahimzadeh‐Vesal, ). The optimal concentration of retinoic acid needed to achieve this outcome, however, depends on the type of stem cells studied.…”

“…Fut4 encodes a cell‐surface marker (SSEA‐1), so it can also be used for sorting PGCs. STRA8 is a possible pre‐meiotic marker of male germ cells (Oulad‐Abdelghani et al, ), and previous studies showed that male germ cells obtained from Stra8 ‐positive cells could fertilize mouse eggs (Nayernia et al, ; Nayernia, Nolte, ), so we used this marker to sort differentiated pre‐meiotic male germ cells (Nourashrafeddin, Aarabi, ; Nourashrafeddin, Ebrahimzadeh‐Vesal, ). DDX4 is another germ cell marker expressed throughout the pre‐meiotic to post‐meiotic phases as well as from the spermatogonia to spermatid stages in the human and mouse testis (Castrillon, Quade, Wang, Quigley, & Crum, ; Fujiwara et al, ; Yamauchi et al, ).…”

“…For example, Nayernia, Nolte () simultaneously used a pre‐meiotic reporter ( Stra8 ‐ GFP ) and a post‐meiotic reporter ( Prm1 ‐ DsRed ) to purify differentiated cells based on their stage of maturation during the differentiation of ESCs into sperm cells. We used the same dual‐reporter system and fluorescence‐activated cell sorting to study the various stages of spermatogenesis (Nourashrafeddin, Aarabi, , Nourashrafeddin, Ebrahimzadeh‐Vesal, ). Although such a method allows for separating those differentiated cells with more potential to become mature sperm, the genetic manipulation prevents the translation of these harvested sperm to clinical infertility treatment.…”