Depletion of high-mobility group box 2 causes seminiferous tubule atrophy via aberrant expression of androgen and estrogen receptors in mouse testis

Abstract

High-mobility group box 2 (HMGB2), a chromatin-associated protein that interacts with DNA, is implicated in multiple biological processes, including gene transcription, replication, and repair. HMGB2 is expressed in several tissues, including the testis; however, its functional role is largely unknown. Here, we elucidated the role of HMGB2 in spermatogenesis using HMGB2 knock-out (KO) mice. Paraffin-embedded testicular tissues were obtained from 8-week-old and 1-year-old wild-type and KO mice. Testis weight and number of seminiferous tubules were decreased, whereas atrophic tubules were increased in HMGB2-depleted mice. Immunohistochemistry revealed that atrophic tubules contained Sertoli cells, but not germ cells. Moreover, decreased cell proliferation and increased apoptosis were demonstrated in HMGB2-depleted mouse testis. To elucidate the cause of tubule atrophy, we examined the expression of androgen and estrogen receptors (AR, ERs, respectively), and the results indicated aberrant expression of AR and ERα in Sertoli and Leydig cells. Southwestern histochemistry detected decreased estrogen response element–binding sites in HMGB2-depleted mouse testis. Expression of HMGB1, which has highly similar structure and function as HMGB2, was examined by immunohistochemistry and western blotting, which indicated increased expression in aged HMGB2 KO mouse testis, especially in spermatocytes. These findings indicate a compensatory increase in HMGB1 expression in HMGB2 KO mouse testis. In summary, depletion of HMGB2 induced aberrant expression of AR and ERα, leading to decreased germ cell proliferation and increased apoptosis that resulted in focal seminiferous tubule atrophy.

An Advanced Detection System for <i>In Situ</i> Hybridization Using a Fluorescence Resonance Energy Transfer-based Molecular Beacon Probe

“…The experimental protocol was approved by the Animal Ethics Review Committee of the University of Miyazaki (#2012-502-3). After animals were sacrificed, tissues were obtained and fixed in 4% PFA in PBS (pH 7.4) at room temperature for 24 hr and then embedded in paraffin [35].…”

Pivotal role of High-Mobility Group Box 2 in ovarian folliculogenesis and fertility

“…In our recent study, we demonstrated that HMGB2 is expressed in male germ cells, whereas HMGB1 is expressed in Sertoli cells. However, a compensatory increase in HMGB1 was detected in germ cells of HMGB2-KO mouse testis [ 11 ]. Therefore, the functional relationship between HMGB1 and HMGB2 requires further clarification in the mouse ovary.…”

Spatiotemporal expression of HMGB2 regulates cell proliferation and hepatocyte size during liver regeneration

“…After washing with 0.075% Brij L23 in PBS, the sections were reacted with HRP-goat anti-mouse IgG or HRP-goat anti-rabbit IgG for 1 h. After washing in 0.075% Brij L23 in PBS, the HRP-sites were visualized with FITC-conjugated tyramide, then microwaved at 95ºC for 15 min in 10 mM citrate buffer (pH 6.0). Then, next primary antibody was reacted for overnight and repeated its detection with rhodamine conjugated tyramide and then counterstained with DAPI 12 , 41 . In some experiments, HNF-4α was visualized by Alexa-633 conjugated goat anti-rabbit IgG.…”

“…Liver tissues were homogenized in hot SDS lysis buffer with a glass-teflon homogenizer, as described previously 12 , 47 . After centrifugation of the homogenate at 15,000 rpm for 30 min at 4 °C, the supernatant was collected and stored at − 80 °C.…”

“…HMGB2 is involved in the fine-tuning of gene transcription 9 , and can bend DNA by loosening wrapped DNA and enhance the accessibility of transcription factors and direct protein–protein interactions with specific transcription factors 10 . HMGB2 is highly expressed in all tissues during embryogenesis, but is preserved in only a few tissues in adults, such as testis, ovary and lymphoid tissue 11 , 12 . HMGB2 is overexpressed in highly proliferative tissues such as testis, ovary, various cancers, and downregulated in senescent cells and aging tissues 13 – 17 .…”