Effects of Acetaminophen on Reproductive Activities in Male Golden Hamsters.

Acetaminophen [Paracetamol, N-acetyl-para-aminophenol (APAP)] is a common over-the- counter analgesic agent as nonsteroidal anti-inflammatory drugs (NSAIDs). The high doses or the long-term treatment of acetaminophen via usual gavage feeding resulted in damage of testicles that presented recoverable impairment, as well as liver and kidney. The influence of acetaminophen was examined in male golden hamsters treated with acetaminophen- containing diet feeding. They were divided into 5 groups and subjected to this experiment for 4 weeks: animals housed in long photoperiod (LP) as LP control, animals housed in short photoperiod (SP) for 4 weeks as SP control (SP4), and groups of animals treated with low, middle, and high concentrations of acetaminophen (Low, Middle, High groups). Also animals housed in SP for 8 weeks were included (SP8) to contrast testicular activities, if necessary. As results, spermatozoa filled the seminiferous tubules of the testicles of animals in LP control and SP4 groups. The aspects were seen in the animals taken diets of low and middle doses of acetaminophen. The animals who fed high dose of acetaminophen showed large or small testicles. The large testicles displayed all germ cells at the steps of spermatogenesis. The small testicles presented no sperm as the animals housed in SP for 8 weeks. Thus these results indicate that acetaminophen invokes the antigonadal effects and accelerates the regressing process of the testicles in the animals compared to the animals exposed to SP.

Transforming Stimulated Clone 22 (TSC-22) Interacts Directly with Bromodomain-Containing Protein 7 (BRD7) to Enhance the Inhibition of Extracellular Signal-Regulate Kinase (ERK) Pathway in Ovarian Cancer.

Bromodomain-containing protein 7 (BRD7) participates in many cellular processes and embryo development. BRD7 is down-regulated in various cancers and evidence of its tumor suppressor function has been accumulating. Here, we identified transforming stimulated clone 22 (TSC-22) as a novel BRD7 interacting protein and show its novel function as a positive regulator of BRD7. We found that TSC-22 expression potentiated the inactivation of the extracellular signal-regulate kinase (ERK) pathway by BRD7. Our data establishes TSC-22 as a modulator of BRD7 and unravels the molecular mechanisms that drive the synergistic tumor-suppressing effects of TSC-22 and BRD7. Our findings may open new avenues for developing novel molecular therapies for tumors exhibiting down-regulated BRD7 and/or TSC-22.

Expression of Kisspeptin in the Adult Hamster Testis.

Kisspeptins, products of KISS1 gene, are ligands of the G-protein coupled receptor (GPR54), and the kisspeptin-GPR54 signaling has an important role as an upstream regulator of gonadotropin releasing hormone (GnRH) neurons. Interestingly, extrahypothalamic expressions of kisspeptin/GPR-54 in gonads have been found in primates and experimental rodents such as rats and mice. Hamsters, another potent experimental rodent, also have a kisspeptin-GPR54 system in their ovaries. The presence of testicular kisspeptin-GPR54 system, however, remains to be solved. The present study was undertaken to determine whether the kisspeptin is expressed in hamster testis. To do this, reverse transcription-polymerase chain reactions (RT-PCRs) and immunohistochemistry (IHC) were employed. After the nest PCR, two cDNA products (320 and 280 bp, respectively) were detected by 3% agarose gel electrophoresis, and sequencing analysis revealed that the 320 bp product was correctly amplified from hamster kisspeptin cDNA. Modest immunoreactive (IR) kisspeptins were detected in Leydig-interstitial cells, and the weak signals were detected in germ cells, mostly in round spermatids and residual bodies of elongated spermatids. In the present study, we found the kisspeptin expression in the testis of Syrian hamster. Further studies on the local role(s) of testicular kisspeptin are expected for a better understanding the physiology of hamster testis, including photoperiodic gonadal regression specifically occurred in hamster gonads.

Expressions of Gonadotropin Subunit Genes in the Reproductively Inactive Golden Hamsters.

Photoperiod has well been established to regulate testicular activities in golden hamsters. These animals breed actively around summer but become infertile in winter. In males, testicles are full of multistep germ cells including spermatozoa in summer. But in winter only fundamental cells consisting of the testicles are detected. The testicular degeneration is accompanied by the reduced levels of blood gonadotropins and testosterone. In this study, the expressions of gonadotropin subunit genes were investigated in the reproductive active and inactive testicles. And parts of sequences of the gonadotropin subunits were identified and compared with those of other rodents. As results, common gonadotropin alpha (CGa), follicle-stimulating hormone (FSH) ß, and luteinizing hormone (LH) ß genes were equivalently detected in pituitaries of both sexually active and inactive animals. In considering low concentrations of gonadotropin hormones determined in pituitary, the present findings imply that the processes involved in translation and/or formation of functional hormones could be impeded in the sexually inactive hamsters. All the nucleotide sequences of gonadotropin subunits identified in this study were same as those reported previously except for one base in CGa. An unsure amino acid deduced from the CGa sequence was confirmed from mRNA sequencing. The outcomes mentioned above suggest that animals with regressed testes prepare for the sexually active period forthcoming in the future.

Blood-Testis Barrier and Sperm Delayed in the Cauda Epididymis of the Reproductively Regressed Syrian Hamsters.

The Syrian (golden) hamsters are seasonal breeders whose reproductive functions are active in summer and inactive in winter. In experimental facility mimicking winter climate, short photoperiod (SP) induces gonadal regression. The blood-testis barrier (BTB) of the sexually involuted animals have been reported to be permeable, allowing developing germ cells to be engulfed or sloughed off the epithelium of the seminiferous tubules. The expressions of genes related to the tight junction composing of BTB were investigated in the reproductive active and inactive testes. Claudin-11, occludin, and junctional adhesion molecule (JAM) were definitely expressed in the active testes but not discernably detected in the inactive testes. And spermatozoa (sperm) were observed in the whole lengths of epididymides in the active testes. They were witnessed in only cauda region of the epididymides but not in caput and corpus regions in animals with the inactive testes. The results imply that the disorganization of BTB is associated with the testicular regression. The developing germ cells are swallowed into the Sertoli cells or travel into the lumen, as supported by the presence of the sperm delayed in the last region of the epididymis. These outcomes suggest that both apoptosis and desquamation are the processes that eliminate the germ cells during the regressing stage in the Syrian hamsters.

A Chronic-Low-Dose Exposing of DEHP with OECD TG 443 Altered the Histological Characteristics and Steroidogeic Gene Expression of Adrenal Gland in Female Mice.

Phthalates and their metabolites are well-known endocrine disrupting chemicals. Di-(2-ethylhexyl) phthalate (DEHP) has been widely used in industry and the exposing possibility to adult is high. In this study, DEHP was treated (133 µg/L and 1,330 µg/L in drinking water) according to the OECD test guideline 443 to mature female mice and their adrenal gland were examined for histological characteristics and steroidogenic gene expression. The wet weight of the adrenal gland was increased in all administrated groups compared to control. The diameter of zona fasciculata (ZF) was increased by DEHP in both outer ZF and inner ZF but there was no difference in morphology of the cells and arrangements into zona between groups. In addition, the arrangement of extracellular matrix was not different between control and DEHP groups. CYP11B1 was mainly localized at ZF and the intensity was not different between groups. DAX1 was localized in zona glomerulosa (ZG) and ZF, and its expression levels were decreased by DEHP administration. Its level was lower in DEHP133 group than DEHP1330 group. On the other hand, CYP17A1 was localized in ZG of DEHP1330 group. These results suggest that chronic low-dose DEHP exposing may modify the microstructure and function of the adrenal cortical cortex.

Histological Analysis of Reproductive System in Low-Dose Nonylphenol-treated F1 Female Mice.

Previously, we reported adverse effects of low-dose nonylphenol (NP) exposure on the reproductive parameters of F1 female mice. In the present study we further investigated the pathohistological effect of NP exposure on the reproductive organs in F1 female mice. NP exposures were continuously conducted from parental pre-mating period until the postnatal day (PND) 33 of F1 offspring for vaginal examination. Mice were sacrificed on PND 30 and the reproductive tissue weights were measured. The initial (at PND 21) body weights of the NP-50 group animals were significantly lower than those of control group animals, and the weight deficit were recovered when the terminal (PND 33) body weights were measured. Early vaginal opening was found in NP group animals (p<0.05). Pathohistological studies revealed that NP-treated F1 animals showed prominent increase in the ovarian follicle numbers (p<0.01), and decrease in the diameter of uterine myometrium (p<0.01), and increase in the diameter of luminal epithelium (p<0.05). The present study demonstrated that the subchronic low-dose NP exposure induced early beginning of puberty and pathohistological abnormalities in ovary and uterus of F1 mice. Further studies are needed to achieve a better understanding on the action mechanism of NP in pubertal onset and to find a way to avoid a hazardous situation provoked by NP exposure.

YY1 and CP2c in Unidirectional Spermatogenesis and Stemness.

Spermatogonial stem cells (SSCs) have stemness characteristics, including germ cell-specific imprints that allow them to form gametes. Spermatogenesis involves changes in gene expression such as a transition from expression of somatic to germ cell-specific genes, global repression of gene expression, meiotic sex chromosome inactivation, highly condensed packing of the nucleus with protamines, and morphogenesis. These step-by-step processes finally generate spermatozoa that are fertilization competent. Dynamic epigenetic modifications also confer totipotency to germ cells after fertilization. Primordial germ cells (PGCs) in embryos do not enter meiosis, remain in the proliferative stage, and are referred to as gonocytes, before entering quiescence. Gonocytes develop into SSCs at about 6 days after birth in rodents. Although chromatin structural modification by Polycomb is essential for gene silencing in mammals, and epigenetic changes are critical in spermatogenesis, a comprehensive understanding of transcriptional regulation is lacking. Recently, we evaluated the expression profiles of Yin Yang 1 (YY1) and CP2c in the gonads of E14.5 and 12-week-old mice. YY1 localizes at the nucleus and/or cytoplasm at specific stages of spermatogenesis, possibly by interaction with CP2c and YY1-interacting transcription factor. In the present article, we discuss the possible roles of YY1 and CP2c in spermatogenesis and stemness based on our results and a review of the relevant literature.

The Effects of Daily Melatonin Gavage on Reproductive Activity in the Male Syrian Hamsters.

The proper administration of melatonin has well been documented to induce testicular regression in seasonal breeding animals. The subcutaneous injections of melatonin in the afternoon, not in the morning, consistently occurred testicular involution in the male Syrian (golden) hamsters whose reproductive activity is regulated by the photoperiod. But the effects of daily melatonin via gavage have not been estimated. Golden hamsters housed in long photoperiod (LP) were divided into 5 groups: the control animals housed in LP or in short photoperiod (SP) and animals treated daily with low (15 µg), middle (150 µg), and high dosages (1,500 µg) of pure melatonin by using gavage in the evening for 8 weeks. As results, LP control animals had large testes and SP controls displayed small and entirely regressed testes. The animals treated with various dosages of melatonin showed collectively degenerating effects on the weights of testes, epididymides, and seminal vesicles in the middle and high dosage groups, with the individual differences as well. The high dosages induced testicular regression in more proportion than the middle dosages did. The low dosage had large testes like the LP control animals. The small and inactive testes shown in some animals of both middle and high groups presented the complete regression as those of the animals maintained in SP. These results strongly suggest that the administrations of melatonin lead to testicular involution in the male golden hamsters when it is administered through gavage.

Adverse Effect of Nonylphenol on the Reproductive System in F2 Male Mice : A Qualitative Change?

Previously, we reported negative effects of low-dose nonylphenol (NP) exposure on the reproductive organs of F1 male mice. In the present study was further investigated the endocrine disrupting effect of NP exposure to F2 generation male mice. Mice were divided into 2 groups; (1) CON, control animals and (2) NP-50 (50 µg/L), animals were treated with NP via drinking water. NP exposures were continuously conducted from parental pre-mating period until the postnatal day (PND) 55 of F2 offsprings. Mice were sacrificed on PND 55 and the reproductive tissue weights were measured. The initial (at PND 21) and terminal (PND 55) body weights of the NP-50 group animals were not significantly different from those of control group animals. NP exposure fail to induce a significant weight change of the testes, seminal vesicle and prostate except absolute epididymal weight (p<0.05). However, pathohistological studies revealed that NP-treated F2 animals showed evident decrease in seminiferous tubule diameters, reduced luminal area and number of germ cells. Also, sloughing morphologies in the tubules were notable. In the caudal epididymis, fewer mature sperms and swollen epithelial cells were found in the NP-treated group. The present study demonstrated that the subchronic low-dose NP exposure induced pathohistological abnormalities in testis and epididymis of F2 mice, and we assumed that these 'qualitative' changes in reproductive tissues could be derived from the epigenetic modifications such as DNA methylation, histone modification, altered DNA accessibility and chromatin structure. Further studies are needed to achieve a better understanding on the multi- or trans-generational effects of NP on the reproductive health and a human application.

Chronic and Low Dose Exposure to Nonlyphenol or Di(2-Ethylhexyl) Phthalate Alters Cell Proliferation and the Localization of Steroid Hormone Receptors in Uterine Endometria in Mice.

Based on our preliminary results, we examined the possible role of low-dose and chronic-exposing of the chemicals those are known as endocrine disrupting chemical (EDC), on the proliferation of uterine endometrium and the localization of steroid receptors. Immunohistochemical or immunofluorochemical methodology were employed to evaluate the localization of antigen identified by monoclonal antibody Ki 67 protein (MKI67), estrogen receptor 1 (ESR1), estrogen receptor 2 (ESR2), and progesterone receptor (PGR). In 133 µg/L and 1,330 µg/L di(2-ethylhexyl) phthalate (DEHP) and 50 µg/L nonylphenol (NP) groups, the ratio of MKI67 positive stromal cells was significantly increased but not in 500 µg/L NP group. The ratios of MKI67 positive glandular and luminal epithelial cells were also changed by the chronic administration of NP and DEHP in tissue with dose specific manner. ESR1 signals were localized in nucleus in glandular and luminal epithelia of control group but its localization was mainly in cytoplasm in DEHP and NP administered groups. On the other hand, it was decreased at nucleus of stromal cells in 1,330 µg/L DEHP group. The colocalization patterns of these nuclear receptors were also modified by the administration of these chemicals. Such a tissue specific and dose specific localization of ESR2 and PGR were detected as ESR1 in all the uterine endometrial tissues. These results show that the chronic lows-dose exposing of NP or DEHP modify the localization and colocalization of ESRs and PGR, and of the proliferation patterns of the endometrial tissues.

Effects of Dietary Supplement Containing Melatonin on Reproductive Activity in Male Golden Hamsters.

Melatonin is a pineal hormone that is synthesized and released at night under the light and dark cycles of a day. Its effects on the reproductive activities have well been established by the administration through various routes in photoperiodic animals. It was also identified in plants and named phytomelatonin. The capacity of the phytomelatonin was investigated in this investigation whether it affects the reproductive function in male golden hamster. As expected, animals housed in long photoperiod (long photoperiod, LP>12.5 hours of lights in a day) had large testes and animals kept in short photoperiod (SP≦12.5 hours of lights in a day) showed remarkably reduced testes. The dietary supplement with melatonin itself induced the complete involution of testes. Pistachios that were reported to contain a large amount of melatonin demonstrated no effects at all in male golden hamsters. These results suggest that dietary supplement containing melatonin-rich foodstuff used in this investigation may not be enough to affect the reproductive endocrine system in male golden hamsters.

Expressions of Semenogelin Gene in Male Syrian Hamsters according to Photoperiod.

The morphogenetically matured spermatozoa (sperm) are generated in the testes by the spermatogenesis. They travel male reproductive tract with many substances secreted from the accessory reproductive organs. One of the substances is the semenogelin (SEMG) released from the seminal vesicles that is involved in the post-testicular maturation. The expression of SEMG gene was investigated in seminal vesicle tissues of sexually matured and regressed male Syrian hamsters by reverse transcription polymerase chain reaction (RT-PCR). The SEMG gene was uniquely identified in the seminal vesicles of the matured Syrian hamsters and compared to the genes reported previously. But the expression of SEMG gene was not observed in reproductively and completely regressed testes of Syrian hamsters. These results indicate that the expressions of the SEMG gene are related to the reproductive capability in the male Syrian hamsters.

The Expressional Pattern of Epididymal Protease Inhibitor (EPPIN) in the Male Syrian Hamsters.

The spermatogenesis is the process by which spermatozoa are generated in the testes. The spermatozoa travel male reproductive tract during which they meet many substances secreted from reproductive organs. One of the substances is epididymal protease inhibitor (EPPIN) that is involved in the post-testicular maturation including capability of fertilizing the eggs. The expression of EPPIN gene was investigated in various tissues of sexually mature and regressed male Syrian hamsters by reverse transcription polymerase chain reaction (RT-PCR). The EPPIN gene was identified in the testis and epididymis of the male Syrian hamsters and compared to the genes reported previously. There was no expression of EPPIN gene in reproductively and completely regressed testes of Syrian hamster. These results suggest that the expressions of the EPPIN gene are associated with the reproductive capability in the Syrian hamsters.

Evolutionary Viewpoint on GnRH (gonadotropin-releasing hormone) in Chordata - Amino Acid and Nucleic Acid Sequences.

GnRH (gonadotropin-releasing hormone) is a supreme hormone regulating reproductive activity in most animals. The sequences of amino acid and nucleic acid of GnRH reported up to now are examined from the evolutionary framework of Chordata. All identified GnRH are classified into GnRH1, GnRH2, or GnRH3. In all three forms of GnRH both N-terminal and C-terminal are conserved, which allows for effective binding to their receptors. The three amino acids in the middle of GnRH1 sequence have altered diversely from the primitive Chordata, which is indicative of the adaptation process to the ambient environment. GnRH2 and GnRH3 sequences are well conserved. There are more diverse modifications in the nucleic acids than in amino acid sequence of GnRH1. These variations can result from meiosis, mutation, or epigenetics and indicate that GnRH is the product of natural selection.

Bitter Melon Seed Extract does not Alter Photoperiodic Effects on Reproduction of Male Golden Hamsters.

Bitter melon (Momordica charantia, MC) has been used in traditional Korean medicine in treating diabetes. In addition, some reports were emerged, showing the antifertility activities of MC in mammals. We investigated the effects of ethanolic MC extract on the reproductive activity of golden hamsters whose spermatogenetic capacity is controlled by their photoperiods. The animals were divided into 4 groups: long photoperiod (LP) control, short photoperiod (SP) control, and LP animals treated with MC. The animals were orally ingested with low (0.03 g/kg) or high (0.15 g/kg) concentrations of the ethanolic extracts for 8 weeks on the daily basis. The control animals received the vehicle. The animals were then mated with age-matched females, experienced pregnancy. As results, the LP control animals showed active large testes but SP control animals displayed remarkably reduced testes. The animals treated with both concentrations of MC extracts demonstrated large testes, indicating fertile activity as animals in LP. LP control animals had litters as expected, but SP controls had no litters at all. MC extract showed the same results as LP animals in generating offsprings. These results suggest that the MC extract does not change the photoperiodic influence on reproductive activity of male golden hamsters.

Hershberger Assays for Bisphenol-A and Its Substitute Candidates.

Bisphenol-A(BPA) is a member of alkylphenol family, and shows adverse effects including reduced fertility, reproductive tract abnormalities, metabolic disorder, cancer induction, neurotoxicity and immunotoxicity. In the present study, we conducted Hershberger assay to evaluate whether the two candidates to replace BPA have androgenic or antiandrogenic activity. The assay was carried out using immature castrated Sprague-Dawley male rats. After 7 days of the surgery, testosterone propionate (TP, 0.4 mg/kg/day) and test materials (low dose, 40 mg/kg/day; high dose, 400 mg/kg/day) were administered for 10 consecutive days by subcutaneous (s.c.) injection and oral gavage, respectively. Test materials were BPA, isosorbide (ISO) and cyclohexanedimethanol (CHDM). The rats were necropsied, and then the weights of five androgen-dependent tissues [ventral prostate, seminal vesicle, levator ani-bulbocavernosus (LABC) muscle, paired Cowper's glands, and glans penis] and three androgen-insensitive tissues (kidney, spleen and liver) were measured. All test materials including BPA did not exhibit any androgenic activity in the assay. On the contrary, antiandrogen-like activities were found in all test groups, and the order of the intensity was CHDM > BPA > ISO in the five androgen-sensitive tissues. There was no statistical difference between low dose treatment and high dose treatment of BPA group as well as ISO group. In CHDM group, high dose treatment exhibited most severe weight reduction in all measured tissues. There was no statistical difference in androgen-insensitive tissue measurements, except BPA groups. Since the effects of ISO treatment on the accessory sex organs were much less or not present at all when compared to those of BPA, ISO could be a strong candidate to replace BPA. CHDM treatment brought most severe weight reduction in all of androgen-sensitive tissues, so this material should be excluded for further screening of BPA substitute selection.

Erratum to : Bitter Melon Seed Extract does not Alter Photoperiodic Effects on Reproduction of Male Golden Hamsters.

[This corrects the article DOI: 10.12717/DR.2017.21.2.215.].

PACAP inhibits tumor growth and interferes with clusterin in cervical carcinomas.

Secretory clusterin (sCLU), an anti-apoptotic protein, is overexpressed in many tumors and enhances tumorigenesis and chemo-resistance. However, the regulation mechanism controlling the sCLU maturation process or activity remains undetermined. In this study, we found PACAP as a negative regulator of CLU. Overexpression of the PACAP gene in cervical cancer cell lines lacking PACAP expression significantly inhibited cell growth and induced apoptosis. We further demonstrated that interaction of PACAP with CLU significantly downregulated CLU expression and secretion, inhibited the Akt-Raf-ERK pathway, and suppressed the growth of human tumor xenografts in nude mice. This novel inhibitory function of PACAP may be applicable for developing novel molecular therapies for tumors with increased sCLU expression.

The Potential Regressive Role of Syzygium aromaticum on the Reproduction of Male Golden Hamsters.

The flower buds of Syzygium aromaticum (clove) have been used as traditional medicine for the treatment of male sexual disorders in Asian countries. Recently, there are some reports about the effects of the clove on reproductive activities in mammals. Therefore, its effect on testicular function was examined in male golden hamsters whose reproductive activity is inhibited by photoperiod such as winter climate. The male animals were given by daily oral administrations (56 consecutive days) in three doses (4 mg, 20 mg, and 100 mg/kg BW) of the alcoholic extract of the clove. Generally lower dose (4 mg) of the extract continued to keep the reproductive activities of testes. The both middle and high doses (20 mg and 100 mg) of the extract completely inhibited the testicular activity in some animals. Taken together, these results suggest a possible biphasic action of alcoholic extract of Syzygium aromaticum flower bud on testicular function.