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.

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Background High-Mobility Group Box 1 (HMGB1) and HMGB2 are chromatin-associated proteins that belong to the HMG protein family, and are involved in the regulation of DNA transcription during cell differentiation, proliferation and regeneration in various tissues. However, the role of HMGB2 in ovarian folliculogenesis is largely unknown. Methods We investigated the functional role of HMGB1 and HMGB2 in ovarian folliculogenesis and fertilization using C57BL/6 wild type (WT) and HMGB2-knockout (KO) mice. Ovarian tissues were obtained from WT and HMGB2-KO mice at postnatal days 0, 3, 7, and 2, 6 months of age, then performed immunohistochemistry, qPCR and Western blotting analyses. Oocyte fertilization capability was examined by natural breeding and in vitro fertilization experiments. Results In HMGB2-KO mice, ovary weight was decreased due to reduced numbers of oocytes and follicles. Natural breeding and in vitro fertilization results indicated that HMGB2-KO mice are subfertile, but not sterile. Immunohistochemistry showed that oocytes expressed HMGB2, but not HMGB1, in neonatal and adult WT ovaries. Interestingly, in HMGB2-KO ovaries, a compensatory increase in HMGB1 was found in oocyte nuclei of neonatal and 2-month-old mice; however, this was lost at 6 months of age. Conclusions The depletion of HMGB2 led to alterations in ovarian morphology and function, suggesting that HMGB2 plays an essential role in ovarian development, folliculogenesis and fertilization.

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