Olfactory receptors (OR) are primarily responsible for the detection of odorant molecules. We previously demonstrated that OR7D4, an OR for androstenone, is expressed in the vomeronasal organ and olfactory epithelium tissue of stallions. Recently, the expression of OR1I1 in the human testes was reported and the possible roles of OR1I1 in the testicular cells were suggested. The objectives of this study were 1) to explore the expression of OR7D4 and OR1I1 in stallion testes, and 2) to define the specific localization of OR7D4 and OR1I1 in the testicular tissues. Stallion testicular tissue samples were used for this study. Western blot was performed to confirm the cross-reactivity of OR7D4 and OR1I1 antibody with stallion testicular tissue samples. OR7D4 and OR1I1 gene expressions were investigated using reverse transcriptionpolymerase chain reaction (RT-PCR) in stallion testes. Immunofluorescence was performed to investigate the expression of OR7D4 and OR1I1 in stallion testicular tissues. The protein bands for OR7D4 and OR1I1 from the testes were observed at approximately 38 kDa and 43 kDa, respectively. The mRNA of OR7D4 and OR1I1 were detected in stallion testes. Immunolabeling of OR7D4 and OR1I1 in the cytoplasm of both spermatogonia and Leydig cells was observed. In conclusion, androstenone and another odorant chemical, which is recognized by OR1I1, may play an important role in stallion testes.

To date, there are no protocols optimized to the effective separation of spermatogonial stem cells (SSCs) from testicular cells derived from mouse testes, thus hindering studies based on mouse SSCs. In this study, we aimed to determine the most efficient purification method for the isolation of SSCs from mouse testes among previously described techniques. Isolation of SSCs from testicular cells derived from mouse testes was conducted using four different techniques: differential plating (DP), magnetic-activated cell sorting (MACS) post-DP, MACS, and positive and negative selection double MACS. DP was performed for 1, 2, 4, 8, or 16 h, and MACS was performed using EpCAM (MACSEpCAM), Thy1 (MACSThy1), or GFR α1 (MACSGFRα1) antibodies. The purification efficiency of each method was analyzed by measuring the percentage of cells that stained positively for alkaline phosphatase. DP for 8 h, MACSThy1 post-DP for 8 h, MACSGFRα1, positive selection double MACSGFRα1/EpCAM, and negative selection double MACSGFRα1/α-SMA were identified as the optimal protocols for isolation of SSCs from mouse testicular cells. Comparison of the purification efficiencies of the optimized isolation protocols showed that, numerically, the highest purification efficiency was obtained using MACSGFRα1. Overall, our results indicate that MACSGFRα1 is an appropriate purification technique for the isolation of SSCs from mouse testicular cells.

Endocrine system of hormones is the critical factor for the development of testes. The levels of hormones are orchestrated by a positive or negative feedback system controlled by the hyphothalamic-pituitary-gonad axis. The aim of this study was to investigate the effect of unbalanced endocrine system induced by the hemi-castration on testicular development in stallions. Four Thoroughbred stallions (age ranging from 3 to 5 yr) were used in this study. To disturb endocrine system, hemicastration has been performed on the stallions. Several parameters including testicular weight, volume, germ cell population on the cross-sections of round tubule, and the area of seminiferous tubules of stallion testes collected at the 1st hemi-castration and the 2nd hemi-castration (about 1 year after 1st hemi-castration) were compared. Testosterone levels were compared for 3 weeks before, after 1st castration, and before 2nd castration using enzyme-linked immunosorbent assay (ELISA) analysis. Immunohistochemistry (IHC) procedure was conducted to compare germ cell populations between after 1st and 2nd castration using VASA antibody. The VASA positive cell population per a cross section of round seminiferous tubule was obtained by monitoring 100 tubules. Interestingly, the weight of testes obtained at 2nd hemi-castration (384±14 g) were significantly higher compared to that of testes collected at the 1st hemi-castration (288±34 g). The volume of testes (306±34 ml) collected at the 2nd hemi-castration was higher than that of testes (169±18 ml) collected at the 1st castration. In contrast, VASA positive germ cell population on the cross section of round tubule (124.9±12.4 vs 142.9±21.6) and the area of round tubule (124±9.7 vs 122.9±1.7 mm2) were not different after 1st castration and 2nd castration. the testosterone levels in the blood collected before, after 1st castration, and before 2nd castration were not significantly different. In conclusion, the hemi-castration induces testicular development to maintain the normal reproductive systems in stallions.

플라스틱 제품의 가소제로 널리 사용되며, 최근 내분비 교란물질로 알려져 있는 di-(2-ethylhexyl)phthalate(DEHP)를 흰쥐에 15일 동안 구강 투여(1g/kg/day, 2g/kg/day, 3g/kg/day)한 후, 정자형성과정에 연관된 정소의 기능과 구조에 미치는 영향을 조사하였다. DEHP 처리군에서는 대조군에 비하여 체중 증가율이 감소하였을 뿐만 아니라 정소의 무게도 감소하였다. 또한 세정관의 직경이 고농도군으로 갈수록 작아지는

Sirt1 belongs to class III histone/protein deacetylase. Sirt1 KO mice have spermatogenic defects. In this study, expression of Sirt1 and acetylated histone 3k9 (H3k9ac) was examined in developing mouse testes. Among two splicing variants of sirt1 mRNA long form was dominant in developing testis. Testicular sirt1 mRNA levels were low at birth, increased until 14 days post partum (pp) and remained constant thereafter. Sirt1 immunoreactivity was weak to negligible in gonocytes, moderate in spermatogonia, absent in preleptotene spermatocytes, moderate in zygotene spermatocytes, strong in pachytene spermatocytes, and weak in diplotene spermatocytes. Round and elongating spermatids were negative for Sirt1. Acetylated histone 3k9 (H3k9ac) immunoreactivity was moderate in spermatogonia and weak to negligible in preleptotene to pachytene spermatocytes but strong in round and elongating spermatids. In Sertoli cells, nuclear Sirt1 immunoreactivity was absent at birth, increased at 14 days pp and markedly increased at 28 days pp onwards. In immature Sertoli cells culture, FSH and testosterone increased sirt1 mRNA, suggesting that Sirt1 participates in protein deacetylation events during the differentiation of Sertoli cells by gonadotropin. In the Leydig cells, nuclear Sirt1 immunoreactivity was weak until 2 weeks pp and decreased in 4 weeks pp onward. suggesting the protein deacetylation by Sirt1 in Leydig cell precursor/progenitor cells. Mutually exclusive expression between Sirt1 and H3k9ac in pachytene spermatocytes in testis suggests that deacetylation of H3K9ac by Sirt1 participates in the gene silencing and/or chromosome behavior in pachytene sspermatocytes.

Mature mammalian oocytes are ovulated at the metaphase II stage of meiosis and complete the cell cycle by fertilization with sperm. During fertilization sperm release an egg activation protein, pho-spholipase C zeta (PLCZ) into oocyte cytoplasm, PLCZ hydrolyze PIP2 into IP3 and DAG. The elevation of IP3 concentration induces Ca2+ release from endoplasmic reticulum (ER) by binding to the IP3 receptor (IP3R) on the membrane of ER. Recent studies have shown that sperm from patients lacking expression of PLCZ1 or expressing mutant forms of PLCZ1 fail to induce [Ca2+]i oscillations or oocyte activation. Purified recombinant human PLCZ1 (hPLCZ1) protein evaluated its [Ca2+]i oscillation activity in mouse and human oocytes. Here we investigated that produced mouse PLCZ-specific antibodyrecognized the PLCZ protein in mouse testes. PLCZ antibody was raised in rabbits against 19-mer sequence at the C-terminus (MENKWFLSMVRDDFKGGKI) of mouse PLCZ protein. Sperm were fixed in 3.7% paraformaldehyde followed by permeabilization. Sperm were incubated in 5% normal goat serum (NGS) and then incubated overnight with anti-mouse PLCZ. Peanut agglutinin (PNA)-lectin was used for detection of the acrosome. Mouse testes from 6~8 weeks old ICR mouse were fixed in 10% formalinand serial sectioned at 5~8um. Testes tissues were immunostained with anti PLCZ antibody and peanut agglutinin(PNA) for acrosome staining. Produced anti mouse PLCZ antibody recognized 74 kDa protein in western and PLCZ is localized to the post-acrosomal region of mouse sperm and to the equatorial region of bull sperm. Mouse PLCZ protein wasdetected on spermatocytes, spermatid, but not on spermatogonia in seminiferous tubules. Some residual bodies on sperm neck and tail showed strong signal of PLCZ, but this staining was still present with antigenic peptide pretreatment to reduce non specific antibody reaction. Also this antibody reacted with the apical region (arrowheads) of principal cells, where secretory vesicles accumulate on the epididymal tissue. But antigenic peptide pretreatment did not remove this apical region staining. This study presents PLCZ protein is localized on the post-acrosomal region or equatorial region of mouse and bull sperm head. Also PLCZ protein in mouse testes expressed from spermatocytes to mature sperm on later stage of spermatogenesis.