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Fas ligand (FasL) and its receptor Fas have been implicated in granulosa cell apoptosis during follicular atresia. Although interferon-gamma (IFN-γ) is believed to be involved in the regulation Fas expression in differentiated granulosa or granulosa-luteal cells, the expression of this cytokine and its role in the regulation of the granulosa cell Fas/FasL system and apoptosis during follicular maturation have not been thoroughly investigated. In the present study, we have examined the presence of IFN-γ in ovarian follicles at different stage of development by immunohistochemistry and related their relative intensities with follicular expression of Fas and FasL, and with differences in granulosa cell sensitivity to Fas activation by exogenous agonistic Anti-Fas monoclonal antibody (Fas mAb). Although IFN-γ immunostaining was detectable in oocyte and granulosa cells in antral follicles, most intense immunoreactivity for the cytokine was observed in these cells of preantral follicles. Intense immunoreactivity for IFN-γ was most evident in granulosa cells of atretic early antral follicles where increased Fas and FasL expression and apoptosis were also observed. Whereas low concentrations of IFN-γ (10-100 U/mL) significantly increased Fas expression in undifferentiated granulosa cells (from preantral or very early antral follicles) in vitro, very higher concentrations (≥ 1,000 U/mL) were required to up-regulate of Fas in differentiated cells isolated from eCG-primed (antral) follicles. Addition of agonistic Fas mAb to cultures of granulosa cells at the two stages of differentiation and pretreated with IFN-γ (100 U/mL) elicited morphological and biochemical apoptotic features which were more prominent in cells not previously exposed to the gonadotropin in vivo. These findings suggested that IFN-γ is an important physiologic intra-ovarian regulator of follicular atresia and plays a pivotal role in regulation of expression of Fas receptor and subsequent apoptotic response in undifferentiated (or poorly differentiated) granulosa cells at an early (penultimate) stage of follicular development.

Background: Bisphenol A (BPA) has been detected in human body fluids, such as serum and ovarian follicular fluids. Several reports indicated that BPA exposure is associated with the occurrence of several female reproductive diseases resulting from the disruption of steroid hormone biosynthesis in the adult ovary. Objective: We hypothesized that long-term exposure to low concentrations of BPA disrupts 17β-estradiol (E2) production in granulosa cells via an alteration of steroidogenic proteins in ovarian cells. Methods: Adult female rats received BPA for 90 days by daily gavage at doses of 0, 0.001, or 0.1 mg/kg body weight. We determined serum levels of E2, testosterone (T), follicle-stimulating hormone (FSH), and luteinizing hormone (LH). We also analyzed the expressions of steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage (P450scc), 3β-hydroxysteroid dehydrogenase isomerase (3β-HSD), and aromatase cytochrome P450 (P450arom) in the ovary. Results: Exposure to BPA significantly decreased E2 serum concentration, which was accompanied by augmented follicular atresia and luteal regression via increase of caspase-3–associated apoptosis in ovarian cells. After BPA exposure, P450arom and StAR protein levels were significantly decreased in granulosa cells and theca-interstitial (T-I) cells, respectively. However, P450scc and 3β-HSD protein levels remained unchanged. The increase in LH levels appeared to be associated with the decreased synthesis of T in T-I cells after BPA exposure via homeostatic positive feedback regulation. Conclusions: BPA exposure during adulthood can disturb the maintenance of normal ovarian functions by reducing E2. The steroidogenic proteins StAR and P450arom appear to be targeted by BPA.

The monogonont rotifer, Brachionus ibericus (S type), is considered to be a promising model species for developmental biology, evolution, and environmental genomics. In an attempt to accelerate the molecular understanding of B. ibericus, we sequenced 680.5 Mb of genomic DNA using the genome sequencer GS-FLX-Titanium. We obtained 2,062,621 reads (average read length 329.9 bp) and 145,418 contigs (total contigs length 125.7 Mb) after excluding small reads (less than 200 bp) from the assembly, and finally obtained 10,133 unigenes (E value ≤ 9.00E−04) after non-redundant (NR) BLAST search. In this article, we summarize the genomic DNA sequences of B. ibericus and discuss their potential use in the study of reproductive biology, endocrinology, environmental genomics, and ecotoxicological studies, and for providing insight into the genetic basis of mechanisms such as egg formation, antioxidant stress defense, and xenobiotic metabolism.

Potential applications of embryonic stem (ES) cells are not limited to regenerative medicine but can also include in vitro screening of various toxicants. In this study, we established mouse ES cell lines from isolated blastomeres of two-cell stage embryos and examined their potential use as an in vitro system for the study of developmental toxicity. Two ES cell lines were established from 69 blastomere-derived blastocysts (2.9%). The blastomere-derived ES (bm-ES) cells were treated with mono-(2-ethylhexyl) phthalate (MEHP) in an undifferentiated state or after directed differentiation into early neural cell types. We observed significantly decreased cell viability when undifferentiated bm-ES cells were exposed to a high dose of MEHP (1000 microM). The cytotoxic effects of MEHP were accompanied by increased DNA fragmentation, nuclear condensation, and activation of Caspase-3, which are biochemical and morphological features of apoptosis. Compared to undifferentiated bm-ES cells, considerably lower doses of MEHP (50 and 100 microM) were sufficient to induce cell death in early neurons differentiated from bm-ES cells. At the lower doses, the number of neural cells positive for the active form of Caspase-3 was greater than that for undifferentiated bm-ES cells. Thus, our data indicate that differentiating neurons are more sensitive to MEHP than undifferentiated ES cells, and that undifferentiated ES cells may have more efficient defense systems against cytotoxic stresses. These findings might contribute to the development of a new predictive screening method for assessment of hazards for developmental toxicity.

Gonadotropin-releasing hormone (GnRH) plays a pivotal role in the regulation of reproduction in vertebrates through interaction with a specific receptor. The GnRH-stimulated gonadotropin synthesis and release are regulated by the GnRH receptors (GnRHRs). In this study, we have identified a GnRH receptor (GnRHR) gene from the hermaphroditic fish Kryptolebias marmoratus. K. marmoratus GnRHR showed typical vertebrate GnRHR domains and motifs, and its cDNA contained 1634 bp including an open reading frame (ORF) of 1263 bp encoding a putative protein of 420 amino acids. To analyze expression patterns of GnRHR gene in various tissues and developmental stages of K. marmoratus, we carried out quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). The K. marmoratus GnRHR gene expression was detected in all the tissues of adult fish with highest level in brain and gonad. The expression of K. marmoratus GnRHR mRNA increased from stage 1 (2 day post fertilization, dpf) to stage 4 (12 dpf) but steeply decreased at hatching stage (stage 5). Expression of K. marmoratus GnRHR after exposure to endocrine-disrupting chemicals such bisphenol A (BPA, 600 microg/L for 96 h) and 4-tert-octylphenol (OP, 300 microg/L for 96 h) in hermaphrodites as well as secondary males was highly up-regulated in almost all the tissues. Another EDC, 4-nonylphenol (NP, 300 microg/L for 96 h) showed no consistent response. 17beta-estrodiol (E2, 100 ng/L for 96 h), a known natural estrogen, suppressed expression of GnRHR in most of the tissues from hermaphrodites as well as secondary males. Tamoxifen (TMX, 10 microg/L), an estrogen antagonist, on the other hand, caused upregulation of GnRHR expression in the liver of hermaphrodites and the gonad and liver of secondary males. This is the first report of a GnRHR gene from K. marmoratus and modulation of its expression by EDCs. This study provides an insight into the molecular mechanism of endocrinological functions of this unique fish.

Tributyltin (TBT) is known to disrupt the development of reproductive organs, thereby reducing fertility. The aim of this study was to evaluate the acute toxicity of TBT on the testicular development and steroid hormone production. Immature (3-week-old) male mice were given a single administration of 25, 50, or 100 mg/kg of TBT by oral gavage. Lumen formation in seminiferous tubule was remarkably delayed, and the number of apoptotic germ cells found inside the tubules was increased in the TBT-exposed animals, whereas no apoptotic signal was observed in interstitial Leydig cells. Reduced serum testosterone concentration and down-regulated expressions of the mRNAs for cholesterol side-chain cleavage enzyme (P450scc), 17α-hydroxylase/C17–20 lyase (P45017α), 3β-hydroxysteroid-dehydrogenase (3β-HSD), and 17β-hydroxysteroid-dehydrogenase (17β-HSD) were also observed after TBT exposure. Altogether, these findings demonstrate that exposure to TBT is associated with induced apoptosis of testicular germ cells and inhibition of steroidogenesis by reduction in the expression of steroidogenic enzymes in interstitial Leydig cells. These adverse effects of TBT would cause serious defects in testicular development and function.

Objective To provide evidence that the Fas-mediated apoptotic process may participate in developing hypospermatogenesis, such as maturation arrest (MA) and Sertoli cell–only syndrome (SCO). Design Prospective clinical and descriptive study. Setting Hospital-based infertility research laboratory and university laboratory. Patient(s) Twenty-two testicular biopsy specimens were obtained for routine clinical purposes from 12 men with nonobstructive azoospermia and from 10 men with obstructive azoospermia. Intervention(s) In situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining was used for detection of apoptosis. Reverse transcription–polymerase chain reaction and immunohistochemical analyses were used for detection of testicular expressions of Fas, Fas ligand (FasL), and caspase-3. Main Outcome Measure(s) Apoptotic indices and testicular expressions of apoptosis regulators. Result(s) Increased apoptosis of germ cells and Sertoli cells was observed in MA and SCO compared with normal spermatogenesis. In testes with MA, increased immunostaining for FasL was observed in the Sertoli cells and Leydig cells, while intense immunostaining of Fas was observed in primary degenerating spermatocytes. Active caspase-3 immunostaining was detected in the cytoplasm of both Sertoli cells and germ cells. In cases of SCO, expression of Fas, FasL, and active caspase-3 was detected both in Sertoli cells and in hyperplastic interstitial cells. Conclusion(s) The current study demonstrates that the expression of FasL is upregulated in the testes of patients with SCO and MA, which suggests that it may be associated with apoptotic elimination or altered maturation of Fas-expressing germ cells through the activation of caspase-3.

4-tert-octylphenol (OP) is known to disrupt testicular development and reduce male fertility. In the present study, male mice were exposed to OP at two different developmental stages, and the expression of steroidogenic enzymes and testosterone production were evaluated. Juvenile (15-day-old) and adult (8-week-old) male mice were injected with 2, 20, or 200 mg/kg of OP or 0.2 microg/kg of estradiol valerate for 5 days. Testosterone concentration was measured by radioimmunoassay and the expressions of the testicular genes were determined by RT-PCR analysis and immunohistochemistry. In the animals exposed with 20 mg/kg of OP during juvenile stage, histochemical analysis of the testis showed that number of pyknotic germ cells inside the tubule was increased, while the number of oil red O positive Leydig cells was decreased. Moreover, the lumen formation was remarkably delayed. A reduced serum testosterone concentration and down-regulated expressions of the mRNAs for steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc), and 17alpha-hydroxylase/C(17-20) lyase (P450(17alpha)) were also observed after juvenile exposure to OP. Immunohistochemical staining for P450scc was mainly detected in interstitial Leydig cells, and a slightly reduced expression of P450scc protein was observed in the testis exposed to 20 mg/kg of OP during juvenile stage. The present study demonstrates that juvenile exposure to OP inhibits steroidogenesis by decreasing the expressions of steroidogenic enzymes in the testis. Diminished lipid content in Leydig cells and reduced transcriptional expression of the cholesterol transport gene, StAR, also support altered cholesterol metabolism and/or transport as a potential mechanism for the decreased testosterone production following exposure to OP. Altogether, the alteration of steroidogenesis by exposure to OP may adversely affect the normal development of the testis and spermatogenesis.

To investigate the impacts of marine pollution on aquatic organisms, we tested the intertidal copepod Tigriopus japonicus as a model species. To analyze the copepods' responses to endocrine-disrupting chemicals (EDCs), we exposed them to two different chemicals: 4,4'-octylphenol (4,4'-OP, 12.5-100 microg/L for 2 h) and polychlorinated biphenyl (PCB, 6.25-25 microg/L for two days). 4,4'-OP was toxic, although exposure time was limited to 2h. After extracting total RNA from the exposed T. japonicus, we performed reverse transcriptase-polymerase chain reaction (RT-PCR) to determine gene expression patterns following chemical exposure. To analyze the gene expression of T. japonicus, we used glutathione S-transferase with GAPDH as an internal control. Of the genes tested using EDC-exposed samples, 4,4'-OP induced upregulation of the glutathione S-transferase (GST) gene, while PCB caused downregulation of the GST gene. These results suggest that the two EDCs act in different manners in T. japonicus.

We cloned two Bombina orientalis ferritin heavy chains (ferritin heavy chains 1 and 2) and one hemoglobin b-chain gene from a B. orientalis oviduct cDNA library, and the length of transcripts was 882, 858 and 611 bp encoding 177, 177 and 148 aa, respectively. B. orientalis ferritin heavy chain genes showed high similarity to those of amphibia (88–93%), mammals (70–71%), and fishes (70–72%), and the hemoglobin b-chain gene showed moderate similarity to amphibian (65–68%) and mammalian (54–57%) hemoglobin b-chain genes, respectively. Based on phylogenetic analysis, the genes were clustered to the same clade in amphibia. The two B. orientalis ferritin heavy chain genes showed different tissue-specific gene expression patterns. Thus, ferritin heavy chain 1 gene was highly expressed in intestine and oviduct but ferritin heavy chain 2 gene was ubiquitously expressed in most of the examined tissues. The hemoglobin b-chain gene was more highly expressed in liver than in oviduct. These findings indicate that the genes may play different roles in different tissues. In this paper, we discuss the basic characteristics of B. orientalis ferritin heavy chain genes and hemoglobin b-chain gene.

To use two small fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) and the Japanese medaka Oryzias latipes (Belloniformes) as testing models in molecular ecotoxicology, we have cloned the cytochrome P450 1A (CYP1A) gene after screening of both genomic DNA libraries, and sequenced 11,863 and 7,243 bp including all the exons and introns with promoter regions, respectively. The Rivulus and the medaka CYP1A gene consisted of seven exons (including non-coding exons) with high homology to mammals. In the promoter region, Rivulus CYP1A gene has seven xenobiotic response elements (XREs) and two metal response elements (MREs), while the Japanese medaka CYP1A gene has six XREs and four MREs. Interestingly, medaka CYP1A gene has a number of MREs at the promoter, which may affect its response on metal exposure. We describe here the gene structure of both fish CYP1A genes.

We have cloned the proto-oncogene c-fos from a self-fertilizing fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) after screening of R. marmoratus lambdaGEM-11 genomic DNA library, and sequenced over 12 kb including all exons, introns and the promoter region. The R. marmoratus c-fos gene consisted of one noncoding exon and four exons with high similarity to those of fugu and mammals. We sequenced approximately 7 kb of the R. marmoratus c-fos gene promoter region to gain a better understanding of the molecular anatomy of the immediate response of this gene upon cellular damage. In the promoter region, R. marmoratus c-fos gene has seven xenobiotic response elements (XREs) and eight metal response elements (MREs) as well as two estradiol (E2), 4 NFkappaB, 2 CarG, 2 prolactin (PRL) motifs and one pit1 site, while the 3'-UTR of this gene contains the estrogen response element (ERE). The seven XRE and eight MRE motifs raise the possibility of its regulation by exposure to environmental pollutants. In this paper, we discuss the gene structure of R. marmoratus c-fos gene and compare its promoter region with those of other organisms' c-fos genes. We propose its potential use in ecotoxicology.

The effects of chronic exposure of 4-tert-octylphenol (OP) on the testicular development of prepubertal male rats were evaluated. 4 weeks old rats were injected with 0.8 microg of estradiol valerate (EV) or 20, 40, or 80mg of OP three times a week for one month. A marked reduction of the size and weight of the testis, epididymis, and seminal vesicle was observed in all the three dosages. Serum testosterone concentration was dramatically decreased while serum LH concentration was increased. Seminiferous tubules were reduced in size and showed no mature spermatozoa or late-stage developing spermatids. In addition, testicular germ cells undergoing apoptosis were obviously increased in all the treated groups. The expression of bcl-xL mRNA was significantly decreased in the OP treated groups, whereas the expressions of bcl-2 and bax mRNA were not significantly changed. In conclusion, these results demonstrate that OP severely reduce the size and/or function of the male reproductive organs due to increased apoptosis of testicular germ cells and the decreased biosynthesis of testosterone.

We cloned the Bombina orientalis adenosine triphosphate (ATP) synthase subunit b gene from a B. orientalis oviduct cDNA library. The transcript was 997 bp long and encoded 250 amino acid residues. It showed high similarity to amphibian (84-85%), mammalian (56-62%) and invertebrate (46-50%) sequences. In phylogenetic analyses, it clustered with other amphibian sequences. This gene was highly expressed in brain, intestine and oviduct but not in muscle and liver. In this paper, we report the basic characteristics of B. orientalis ATP synthase subunit b gene.

Tamoxifen (TAM) is the endocrine therapeutic agent the most widely used in the treatment of breast cancer, and it operates primarily through the induction of apoptosis. In this study, we attempted to elucidate the non-ER mediated mechanism behind TAM treatment, involving the phospholipase C-protein kinase C (PLC-PKC) mediated phospholipase D (PLD) activation pathway, using multimodality methods. In TAM treated MCF7 cells, the PLC and PLD protein and mRNA levels increased. Phosphatidylethanol (PEt) and diacylglycerol (DAG) generation also increased, showing increased activity of PLD and PLCgamma1. Translocation of PKCalpha, from cytosol to membrane, was observed in TAM treated cells. By showing that both PKC and PLC inhibitors could reduce the effects of TAM-induced PLD activation, we confirmed the role of PKC and PLC as upstream regulators of PLD. Finally, we demonstrated that TAM treatment reduced the viability of MCF7 cells and brought about rapid cell death. From these results, we confirmed the hypothesis that TAM induces apoptosis in breast cancer cells, and that the signal transduction pathway, involving PLD, PLC, and PKC, constitutes one of the possible mechanisms underlying the non-ER mediated effects associated with TAM.

In the mammal, melatonin regulates the seasonal and/or circadian rhythm of PRL levels. Since several members of the PRL gene family are expressed during late pregnancy, we investigated the relationship between the expression of placental lactogen (PL)-II-one member of the PRL family- and melatonin, as well as the placental expression of one of the receptors for melatonin, melatonin receptor 1a (Mel 1a ) . Herein we provide the first demonstration that Mel 1a is not only expressed in the rat placenta, but that it is spatially and temporally regulated throughout late pregnancy. In situ hybridization and Northern blot analyses show that Mel 1a mRNA is localized in the rat placenta on gestational day 19, and is mainly restricted to the spongiotrophoblast and trophoblast giant cells. Interestingly, the junctional zone of the placenta at this time showed the strongest gene expression when the tissue was obtained at 16:00 h (daytime) and showed the least expression when it was obtained at 04:00 h (night-time). In contrast, the labyrinth zone showed the strongest expression in tissue obtained at night and showed the least expression in tissue obtained during the day. PL-II gene expression also exhibited a circadian rhythm but the direction of the fluctuation was exactly opposite to that of the Mel 1a gene, such that at night the junctional zone had the strongest expression, while the labyrinth zone had the weakest. In vitro treatment of placental tissue with an melatonin agonist, chloromelatonin, greatly decreased PL-II mRNA levels. That Mel 1a plays a regulatory role in the expression of PL-II in the late-pregnancy rat placenta is strongly suggested by the pattern of its own spatial and temporal expression.

The aim of this study was to investigate expression patterns of the prolactin (PRL) and c‐fos genes by 17β‐estradiol (17β‐E) and stress in the mouse pituitary. In the pituitary, the levels of PRL mRNA were found high with some fluctuation at 30, 60, and 90 min whereas the levels of PRL mRNA were low at 120 min when ovariectomized female mice were injected with 17β‐E or vehicle. PRL mRNA levels began to increase again at 4 h and remained high up to 24 h only in the 17β‐E‐treated mice. The overall changes in c‐fos mRNA by 17β‐E were very similar to those in PRL mRNA in the pituitary. Subsequent study revealed that these high initial levels of PRL and c‐fos mRNAs were caused by stress during injection, not by 17β‐E, since vehicle injection alone into the ovariectomized mice could increase the levels of PRL and c‐fos mRNAs. The stress‐induced elevations of PRL and c‐fos mRNAs were inhibited by bromocriptin, a dopamine agonist, suggesting that the dopaminergic system is involved in the action route of injectio...

The immunocytochemical method was used to identify the existence of voltage‐dependent Ca2+‐channels in mouse follicular oocytes. Three types of voltage‐dependent Ca2+‐channels were shown to exist in the follicular oocytes for the first time, the P/Q‐type Ca2+‐channel, the N‐type Ca2+‐channel, and the L‐type Ca2+‐channel. Among proven Ca2+ ‐channels, distributions of the P/Q‐type Ca2+‐channel and L‐type Ca2+ ‐channel showed localized staining (clustered pattern) on the oolemma. The distribution of the P/Q‐type Ca2+ ‐channel showed all localized staining, and the range of localized staining was from 1 to 8 in staining intensity. As the staining intensity increased from 1 to 8, the number of localized staining decreased. The L‐type Ca2+‐channel are homogeneously stained (29.4%‐54.2%), while some of them (around 28.7%‐44.1%) showed localized staining on the oolemma. However, the rest of them showed no staining at all (17.1%‐ 26.5%). On the contrary, the N‐type Ca2+‐channel showed mostly homogeneous staining, ...

Endocrine disrupting chemicals (EDCs) have been a major concern in the normal reproduction and development of aquatic organisms. In the teleost, steroid hormones are synthesized via the steroidogenesis pathway, and play a key physiological role in the regulation of gonadal sex differentiation. The protogynous hermaphroditic fish, Kryptolebias marmoratus is the only vertebrate capable of reproducing through internal self-fertilization. To uncover the effect of bisphenol A (BPA) on sex differentiation genes on transcription, we investigated the expression patterns of several sex differentiation-related genes such as dax1, dmrt1, mis, sf1, figlα, StAR and wt1 after BPA exposure with controls (E2 and TMX). In response to 17β-estradiol (E2) exposure, a testis-specific gene, dmrt1 mRNA was down-regulated in the gonad of the secondary male but the expression of the female-specific gene, dax1 mRNA was significantly elevated in the brain and gonad. A high level of StAR mRNA was detected in the brain and gonad of both hermaphrodite and secondary males, suggesting that the elevated expression of dax1 and StAR genes would be involved in E2 exposure. As expected, upon BPA exposure, the dmrt1 and MIS mRNA level decreased in both hermaphrodite and secondary males, while the female-specific gene, figlα mRNA level increased in the gonad of both genders. BPA showed an opposite mode of action on the expression of dax1 (induction, P>0.05) and sf1 mRNA (inhibition, P>0.05) in the brain and gonad against both genders. The sensitivity of dax1 to BPA on expression was relatively high in the secondary male. The wt1 mRNA was up-regulated in most tissues except in the liver of BPA-exposed secondary males. Regarding the time course study, the figlα mRNA level increased at 6 h after BPA exposure. In addition, BPA elevated the expression of StAR, dax1, and wt1 mRNA but repressed sf1 mRNA. In this paper, we demonstrated that BPA may modulate the expression of sex differentiation and steroidogenesis pathway genes, and this finding would provide a better understanding on the modulation of transcription upon BPA exposure in steroidogenesis and sex differentiation in the hermaphroditic fish, K. marmoratus.

Leydigcellsofthemammaliantestisproducetestosteroneand support spermatogenesis, and thereby their role in male function is fundamental. Although benzo[a]pyrene (B[a]P) has been known to exhibit carcinogenic, apoptogenic, and endocrine-disrupting activities, its potential signaling system in Leydig cells remains to be discovered. In the present study, using the TM3 Leydig cell line and primary Leydig cells, we showed that Leydig cells do not die by exposure to B[a]P and found that an increased level of X chromosome-linked inhibitor of apoptosis protein may be associated with the antiapoptotic process. The Leydig cells were shown to express p53, but its translational level was extremely low. Although a high level of p53 protein was not necessary for apoptosis induced by B[a]P-7,8-diol-9,10-epoxide (a final B[a]P metabolite) in Leydig cells, the apoptosis of primary Leydig cells appears to be p53 independent. This indicates the lack of p53 function in primaryLeydigcells.Furthermore,Leydigcellswerefoundto retain insignificant levels of endogenous aryl-hydrocarbon receptor and AhR nuclear transporter proteins in nature. Exposure to B[a]P did not result in a significant increase in aryl-hydrocarbon receptor proteins that are required for CYP1A1 transcription. CYP1A1 expression was present in Leydig cells but at levels insufficient to exhibit its activity. Finally, we have demonstrated that overexpression of CYP1A1 in Leydig cells sensitizes the cells to exhibit its activity in the presence of B[a]P and, thus, induction of apoptosis. Together, these results indicate that the deficiency of CYP1A1 activity might be a decisive condition rendering Leydig cells secure from exogenous polycyclic aromatic hydrocarbons such as B[a]P. (Endocrinology 148: 6134–6144, 2007)

Objective To estimate chromosomal imbalances in preimplantation embryos from reciprocal translocation carriers with or without acrocentric chromosomes (Acro-Ch) 13, 14, 15, 21, and 22 in preimplantation genetic diagnosis (PGD) cycles. Design Fluorescence in situ hybridization was applied to PGD cycles for reciprocal translocation carriers. Setting University-based centers for reproductive medicine. Patient(s) Ten and 24 patients of reciprocal translocation with and without Acro-Ch, respectively. Intervention(s) Fluorescence in situ hybridization in biopsied blastomeres. Main Outcome Measure(s) Estimation of meiotic segregation mode in embryos from translocation carriers. Result(s) The proportion of alternative segregation for normal or balanced chromosome contents in preimplantation embryos from PGD cycles in reciprocal translocations without Acro-Ch was significantly higher than that with Acro-Ch (26.0% vs. 14.6%). The proportion of interchange trisomy in 3:1 segregation was significantly lower in reciprocal translocations without Acro-Ch than that with Acro-Ch (4.3% vs. 9.5%). Conclusion(s) This is the first report that the incidence of alternative segregation producing normal or balanced embryos was relatively low in reciprocal translocations associated with Acro-Ch. Our data may be useful to predict the possibility of normal or balanced embryos and to counsel with reciprocal translocation carriers for PGD–fluorescence in situ hybridization cycles.

Ovarian follicular degeneration is accelerated by gamma-radiation. To investigate the precise radiation-induced cellular and molecular biological changes in the ovary, prepubertal mice were whole-body irradiated with 6.94 Gy, which is the 30% of the lethal dose of gamma-radiation using a (60)Co source. At 0, 3, 6, 12, and 24 hr after irradiation, ovarian expression of p53 and p21 mRNA and protein were analyzed by reverse-transcription polymerase chain reaction and immunoblotting, respectively. Immunohistochemical localization of p53 and p21 antigens was also carried out. Immunoreactive p53 and p21 were expressed in the nuclei of granulosa cells, but were not detected on the theca. In control mouse ovaries, p21 was weakly expressed on granulosa but not on the theca cells. In gamma-irradiated mouse ovaries, however, immunoreactive p21 proteins were detected in the nuclei of follicular granulosa cells. After irradiation expression of p53 and p21 mRNA and protein was significantly increased compared to beta-actin. Taken together, these observations suggest that p53 and p21 are actively involved in gamma-radiation-induced follicular degeneration in the prepubertal mouse ovary.

The purpose of this study was to correlate temporal expression of clusterin and apoptosis in androgen-independent human prostate cancer cells (PC-3) treated with 25 microM doxazosin. DNA fragmentation, reverse transcriptase polymerase chain reaction, and terminal transferase-mediated biotinylated 16-desoxy-uridene triphosphate nick-end labeling (TUNEL) assays were used to assess degree of apoptosis and temporal and spatial expression of clusterin mRNA and protein. DNA fragmentation was significant at 48 hours. Clusterin mRNA expression was 3-fold higher than control at 9 hours and was maintained over 48 hours. The TUNEL assay showed increasing percentage of apoptotic cells and presence of clusterin after doxazosin treatment. During doxazosin-induced apoptosis in PC3 cells, clusterin appeared to initially accumulate in the cytoplasm and protect against apoptosis; later, after its transport to the nucleus, clusterin was no longer able to suppress apoptosis.

Previous studies on ras proto-oncogene genes in fish have been focused on chemical-associated carcinogenesis, and the expression of fish ras genes was not well-characterized. We investigated Ki- and Ha-ras genes from the hermaphroditic fish Rivulus marmoratus to understand better their expression patterns in specific tissues, as well as their responses to endocrine-disrupting chemicals such as 4-nonylphenol (4-NP). By investigating expression patterns, we found that the R. marmoratus Ki-ras (Rm Ki-ras) gene showed an alternative splicing event between exons 4A and 4B according to tissue types, which is different from the expression pattern of mammalian Ki-ras genes. In the Rm Ki-ras gene, there were two different expressed types, with exons 1-2-3-4A-4B (long form) and with exons 1-2-3-4B (short form). In the Rm Ki-ras gene, the long form was expressed strongly in the gonad and intestine, and the short form was expressed ubiquitously, except for a low level of expression in the liver. Following 4-NP exposure (300 μg/L), the Rm Ki-ras long form in the liver was significantly expressed, while it was expressed moderately in the ovaries. However, the Rm Ha-ras gene was significantly over-expressed in the brain, while its expression in the gonad was down-regulated. In relation to these modulations after 4-NP exposure, we searched the Rm Ha- and Ki-ras promoter regions and found several ERE-half sites, that may be involved in the modulation of ras gene expression following 4-NP exposure. These genes could be applicable as new biomarker genes for assessing exposure to endocrine-disrupting chemicals (EDCs). Further, this implies the disturbance of ras-dependent signal transduction following EDC exposure.

To uncover the effect of estrogenic chemicals [4-nonylphenol (NP) and bisphenol A (BisA)] on the expression of androgen receptor (AR) and estrogen receptors (ERalpha and ERbeta) in the hermaphroditic fish Rivulus marmoratus, we cloned the full length of the cDNAs encoding AR, ERalpha, and ERbeta from gonadal tissue of R. marmoratus and analyzed the modulation of expression of these genes following exposure to estrogenic chemicals using real-time RT-PCR. R. marmoratus AR, ERalpha, and ERbeta genes showed a high similarity to the relevant fish species on amino acid residues, respectively. Rm-ERalpha and Rm-ERbeta cDNAs included a serine-rich region when compared to other teleost fish ER genes. Tissue-specific expression of Rm-AR and Rm-ERbeta mRNAs in adult hermaphrodite R. marmoratus was high in the gonad, while Rm-ERalpha mRNA was high in the liver based on real-time RT-PCR. In addition, Rm-AR and Rm-ERalpha mRNAs increased along with developmental stage from stage 3 (5 dpf) to hatching, while Rm-ERbeta mRNA increased from stage 2 (2 dpf). To uncover the effect of estrogenic chemicals on R. marmoratus, we exposed the fish to NP (300 microg/l) and BisA (600 microg/l) for 96 h. Significant down-regulation of Rm-AR, Rm-ERalpha, and Rm-ERbeta mRNA was observed in gonadal tissue after exposure to NP but not BisA. In the liver, there were gender differences in gene expression after EDC exposure. These results demonstrate that expression patterns of the Rm-AR, Rm-ERalpha, and Rm-ERbeta genes in the hermaphroditic fish, R. marmoratus, vary according to tissue and developmental stage as well as the specificity of environmental estrogenic chemicals. These genes can be useful as molecular biomarkers in assessing the potential impact of estrogenic compounds using this species as a model system.

To understand the effect of endocrine-disrupting chemicals (EDCs) on cytochrome P450 aromatase (rm-cyp19) gene expression between gender types in the hermaphroditic fish Rivulus marmoratus, we cloned two distinct rm-cyp19 genes using RT-PCR with degenerative primers, obtained full-length cDNAs using 5′- and 3′-RACE-PCR methods, and completely sequenced them. The brain aromatase (rm-cyp19b) cDNA consisted of 2,124 bp including the open reading frame (ORF), which encoded a putative protein of 505 amino acids. The ovarian aromatase (rm-cyp19a) cDNA consisted of 2,075 bp, including the ORF encoding a putative protein of 516 amino acids. Expression patterns of rm-cyp19b and rm-cyp19a mRNAs were investigated in embryos of different developmental stages and in seven different tissues of adult fish. The rm-cyp19b gene in hermaphrodite and secondary male R. marmoratus was predominantly expressed in the brain, while the rm-cyp19a gene was expressed gender-specifically in the gonad. The expression of rm-cyp19b mRNA increased from stage 1 (2d post fertilization) to stage 4 (12d post fertilization) in a developmental stage-dependent manner but steeply decreased in the hatching stage. Compared to the rm-cyp19b gene, the abundance of ovarian aromatase rm-cyp19a transcripts was very low, and its expression was first detected at stage 3 and then decreased gradually to the hatching stage. Alteration of rm-cyp19b and rm-cyp19a gene expression was further analyzed in the brain and gonad by real-time RT-PCR 96 h after EDC exposure in hermaphrodites and secondary males. The brain aromatase rm-cyp19b gene was up-regulated in the brain after 4-nonylphenol (4-NP)-exposure, while the ovarian aromatase rm-cyp19a gene was significantly down-regulated in the gonad. In 300 μg/L 4-tert octylphenol (4-tert-OP), or 600 μg/L bisphenol A-exposed brain and gonad, both rm-cyp19b and rm-cyp19a genes were up-regulated. In the case of secondary males, the rm-cyp19b gene was highly expressed in the 4-NP-exposed brain, while expression of the rm-cyp19a gene was not detected in the gonad. These results indicate that the expression of rm-cyp19a and rm-cyp19b genes is differently modulated according to estrogenic compounds and gender type of R. marmoratus.

We cloned two Bombina orientalis ferritin heavy chains (ferritin heavy chains 1 and 2) and one hemoglobin beta-chain gene from a B. orientalis oviduct cDNA library, and the length of transcripts was 882, 858 and 611 bp encoding 177, 177 and 148 aa, respectively. B. orientalis ferritin heavy chain genes showed high similarity to those of amphibia (88-93%), mammals (70-71%), and fishes (70-72%), and the hemoglobin beta-chain gene showed moderate similarity to amphibian (65-68%) and mammalian (54-57%) hemoglobin beta-chain genes, respectively. Based on phylogenetic analysis, the genes were clustered to the same clade in amphibia. The two B. orientalis ferritin heavy chain genes showed different tissue-specific gene expression patterns. Thus, ferritin heavy chain 1 gene was highly expressed in intestine and oviduct but ferritin heavy chain 2 gene was ubiquitously expressed in most of the examined tissues. The hemoglobin beta-chain gene was more highly expressed in liver than in oviduct. These findings indicate that the genes may play different roles in different tissues. In this paper, we discuss the basic characteristics of B. orientalis ferritin heavy chain genes and hemoglobin beta-chain gene.

The organic thiophosphate, amifostine, is a promising pharmacological compound showing selective protection in many tissues against the toxic side-effects of radiation and cytotoxic drugs. The aim of the present study was to assess the radioprotective effects of amifostine on ovarian follicles. Three-week-old female mice, with or without pretreatment with amifostine, were irradiated with 6.42 Gy of gamma-ray. Reduced proliferation of granulosa cells was verified with BrdU staining and the incidences of follicular degeneration increased in ovarian follicles in the gamma-ray-irradiated mice compared to that of the control or amifostine-treated group. Biochemical changes caused by gamma-irradiation provoked a rise of p53 and Bax protein and a decline of the inactive form in caspase-3 and PARP protein. Caspase-3 and PARP cleaved into active peptides during apoptosis. This process was confirmed by the result of this study, which was that the amount of the stable form decreased immediately after irradiation. In the amifostine treatment group before irradiation, the increased rate of p53 and Bax was suppressed, particularly in the LDs-treated group. The relationship between PARP and caspase-3 levels showed the effect of amifostine exposure before irradiation. In conclusion, amifostine had an inhibitory effect on ovarian programmed cell death induced by gamma-ray, affecting the expression of apoptotic signaling molecules and the level of proliferation of the granulosa cells.

Prepubertal mice were whole-body irradiated with a mean lethal dose (LD50) of gamma-radiation using a 60Co source with a total dose of 7.2 Gy and a dose rate of 12.0 cGy/min. At day 0 before the irradiation and at day 1, 2, and 3 after the irradiation, the ovaries were collected and the morphological changes were assessed. The ratios (%) of atretic or polymorphonuclear leukocytes (neutrophil)-infiltrated follicles in the largest cross sections were calculated. In the early atretic follicle of the control mouse ovary, both apoptotic and mitotic cells were observed and occasionally neutrophils were infiltrated into the follicle cavity. However, in the atretic follicles 2 days post-irradiation, numerous cell fragments, apoptotic cells and bodies, and especially, a number of neutrophils were observed. In the non-irradiated control, the ratios of atretic follicles were 58.0+/-8.6 and 27.3+/-11.2 (mean+/-S.E.M.) in antral and preantral follicles, respectively. The ratios of the number of antral and preantral follicles with one or more neutrophils to the total number of atretic follicles were 29.3+/-12.0. At 2 days post-irradiation, the ratios of atretic follicles were increased to 94.0+/-3.4 and 86.9+/-7.6 in antral and preantral follicles, respectively. The ratios of neutrophil-containing follicles among the atretic one were increased to 65.9+/-11.5 and 57.8+/-15.4 at 2 and 3 days after the irradiation, respectively. Taken together, the present results show that gamma-radiation induces apoptotic and inflammatory degeneration of mouse ovarian follicles. Besides, neutrophils may be involved in the acute atretic degeneration in gamma-irradiated mouse ovarian follicles.

Purpose. To investigate the potential effects of pentobarbital and ketamine on serum concentrations of sex hormones, the present study was performed using pregnant mare serum gonadotropin (PMSG)-primed cyclic female Sprague-Dawley rats. Methods. Pentobarbital sodium (37 mg·kg−1, i. p.) or ketamine-hydrochloride (229 mg·kg−1, i. m.) was injected 2 and 3 days after PMSG treatment. At 0, 1, 2, 3, 4, and 5 days after PMSG treatment, sera were collected by cardiac puncture. The serum concentrations of progesterone (P4), testosterone (T), and estradiol-17β (E2) were determined by radioimmunoassay. Results. The serum concentrations of P4 tended to be lower in both the pentobarbital- and the ketamine-treated groups compared with the control group. Significant differences were found on days 3 and 4 after pentobarbital and on days, 1, 4, and 5 after ketamine administration. Serum concentrations of T were also suppressed in both the pentobarbital and the ketamine-treated groups, whereas E2 concentrations decreased only in the ketamine-treated group. Conclusion. Pentobarbital and ketamine decrease serum sex hormone concentrations in PMSG-primed female rats.

Caffeine is the main psychoactive ingredient of coffee, tea and even colas. This study investigated the radioprotection of caffeine in mice. Mice were irradiated with 6.5 Gy. A caffeinetreated group was administered 80 mg/kg-body weight by an i.p injection at 1 hour before irradiation. At 6 hours after irradiation, the animals were sacrificed for the study. Hormonal analysis was performed with radioimmunoassay on serum. The expression kinetics of the apoptotic genes after irradiation was evaluated with RT-PCR. The weight of body and organ and H and E-stained slides did not show differences between groups. The circulating testosterone significantly decreased in the irradiated group. The expression of Fas antigen, p21, p53, bax and bcl2 related to radiation- induced apoptosis was comparable to that of the caffeine-untreated group. Bax mRNA dramatically increased in the irradiated group, but not in the irradiated caffeine -treated group. The results indicate that caffeine protects an early apoptotic initiation after irradiation.

This study was performed to investigate the effects of Aroclor 1254 (A1254), a commercial polychlorinated biphenyl mixture, on the expression of rat placental prolactin (PRL) family genes and reproductive activity. Placental lactogen-Iv and -II, and prolactin-like protein-A and -C mRNA levels were significantly decreased in the placentas of A1254-treated rats in a dose-dependent manner. The mRNA levels of Pit-1alpha and beta isotypes, which are involved in the regulation of PRL family gene expression, were also decreased in the A1254-treated rat placenta. In the rat placental junctional zone, high-dose A1254 (25 mg/kg B.W.) treatment reduced the number of spongiotrophoblasts, cells in which the PRL family genes are expressed. Finally, maternal exposure to A1254 was shown to have significant toxic effects on reproductive activity, including embryonic and placental growth retardation, delay of parturition, and reduction of the number of pups per litter. The results of the present study indicated that A1254 has an inhibitory effect on PRL family, Pit-1alpha, and beta gene expression in the rat placenta, leading to significant toxic effects on reproductive activity in rats.

Melatonin, secreted by the pineal gland, is involved in the regulation of many physiological functions of various species of animals. In the present study, the expression of gene for melatonin Mel(1a) receptor (MelR) was evaluated in the ovary, hypothalamus, and pituitary according to the developmental stages in female mice. Semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) and in situ PCR techniques were applied. According to the developmental stages, gene for MelR was differently expressed on ovary, hypothalamus, and pituitary. MelR gene was first expressed on pituitary prior to the expression in hypothalamus and ovary. Ovarian MelR gene started to express at birth. Unlike hypothalamic expression of MelR gene which was identified after birth, in pituitary, it was expressed at 16 days post coitum. In the ovary, the expression signal of MelR gene was identified on granulosa cells. However, the signal was not detected in the theca cells. It was weak in the primordial and atretic follicles. Taken together, it can be considered that melatonin has a pivotal role in the folliculogenesis.

The present study deals with the morphological changes of the degenerating primordial and primary follicles induced by gamma-radiation. Prepubertal female mice of 3 weeks old ICR strain were gamma-irradiated with the dose of LD(80(30)) (8.3 Gy). The ovaries were collected at 3, 6 and 12 h after irradiation. The largest cross-sections were prepared by histological semithin sections for microscopical observations. The ratio (%) of normal to atretic follicles decreased with time after the irradiation in primordial follicles and in primary follicles as well. At 6 h after irradiation, the number of degenerated primordial follicles increased. Germinal vesicles disappeared and lipid droplets increased in number. Granulosa cells became round in shape and apoptotic cells started to appear. The ooplasmic membrane was not recognizable. The ratio of normal to atretic primordial follicles in the control group was 62.5. Then it became lower with time after the irradiation. It went down to 51.6, 49.0, 11.1 and 7.1 at 0, 3, 6 and 12 h, respectively. The ratio of normal to atretic primary follicles in the control mouse ovary was 81.3. It was 80.0, 75.0, 45.5 and 33. 3 at 0, 3, 6 and 12 h after irradiation, respectively. It is concluded that the ionizing radiation acutely induces the degeneration of primordial and primary follicles. The pattern of degeneration is one of the following: (1) apoptosis of one or more granulosa cells with a relatively intact oocyte, (2) apoptosis of an oocyte with intact follicle cells, or (3) apoptotic degenerations of both kinds of cells. These results can provide morphological clues for the identification of the degenerating primordial and primary follicles in normal and irradiated mouse ovaries.

Although recent studies have demonstrated that ovarian follicular atresia occurs by apoptosis of granulosa cells, the intracellular signaling pathways involved in apoptotic cell death are still poorly characterized. We examined the role of ceramide as a candidate intracellular mediator of Fas-mediated signaling in cultured granulosa cells. Expression of Fas antigen was demonstrated by Western blot of granulosa cell lysates and immunostaining of cultured granulosa cells. Exposure of granulosa cells to anti-Fas monoclonal antibody (anti-Fas mAb) resulted in significant sphingomyelin hydrolysis, which was accompanied by a progressive increase in endogenous levels of ceramide. The addition of exogenous C6-ceramide induced drastic morphological change, including nuclear fragmentation and typical apoptotic DNA degradation. Furthermore, both anti-Fas mAb and C6-ceramide decreased phospholipase D (PLD) activity and diacylglycerol (DAG) concentrations in a time- or a dose-dependent manner. In addition, treatment with phorbol 12-myristate 13-acetate completely attenuated the ceramide-induced inhibition of PLD activity and partially suppressed ceramide-induced apoptosis. These results indicate that the Fas/ceramide signaling pathway might play a role in granulosa cell apoptosis and suggest that the PLD/DAG pathway might be cross-linked to the Fas/ceramide pathway in apoptotic processes of granulosa cells.

In the present study we have examined the presence of Fas, Fas ligand (FasL), and p53 in rat granulosa cells during follicular development and atresia, especially in relation to the granulosa cell cycle progression and the onset of granulosa cell apoptosis. Fas, FasL, and p53 proteins were immunolocalized, and their contents were determined by Western blotting. Granulosa cell apoptosis was assessed by DNA fragmentation analyses (DNA ladder) and in situ terminal deoxynucleotidyl transferase mediated deoxy-UTP-biotin nick end labeling (TUNEL) as well as by flow cytometry. Ovaries not exposed to gonadotropins (control) consisted predominantly of preantral and early (small) antral follicles, the latter of which were mostly atretic and demonstrated intense TUNEL staining in granulosa cells exhibiting positive immunoreactivities for FasL and Fas. Granulosa cells isolated from these follicles were apoptotic, as evident by clear ladder pattern of DNA fragmentation upon electrophoretic analysis and the high percentage (>10%) of the cell population in the A0 phase of the cell cycle. After gonadotropin treatment, these features completely disappeared during each of the 3 days of follicular growth to the medium to large antral stages. Cell cycle analysis showed significantly higher proportion of the cells in S and G2/M phases compared with controls, which was accompanied by marked decrease in immunoreactivities for Fas, FasL, and p53. By days 4 and 5, widespread atresia and extensive granulosa cell apoptosis were noted in large antral and preovulatory follicles and were coincidental to increased expression of p53 and Fas, but not of FasL, as well as an apparent arrest of granulosa cell G1/S progression, as evident by an increased cell population in G0/G1 and a decrease in the S and G2/M. Granulosa cells from equine CG-primed ovaries exhibited marked increases in p53 and Fas protein contents and apoptosis after adenoviral p53-sense complementary DNA infection in vitro and were more responsive to Fas activation by an agonistic Fas monoclonal antibody challenge. Taken together, these findings are consistent with the well accepted concept that gonadotropin plays a central role as a survival factor in the regulation of granulosa cell Fas/FasL and p53 expression during ovarian follicular development. In addition, the control of granulosa cell apoptosis may involve two consecutive cellular/molecular events: cell cycle arrest at G1/S and exit from G0 into A0 phase, via regulation of the p53 and Fas/FasL death pathways.

In order to observe the morphological and endocrinological changes of the rat and mouse ovarian follicles by gamma-radiation, rats were whole-body irradiated with doses of 3.2 Gy and 8.0 Gy and mice with 2.9 Gy and 7.2 Gy. Sections of ovaria were examined by light microscopy. Concentrations of progesterone, testosterone, and estradiol in ovarian homogenate were determined by radioimmunoassay techniques. Gamma-radiation resulted in the increased percentage of atretic follicles in the groups killed on day 0, day 4, and day 8 after irradiation. The decrease in granulosa cell viability was found in animals killed on day 4 after irradiation. The finding of the high ratio of testosterone to estradiol compared to that of progesterone to testosterone suggests that aromatase activity — steroid biosynthesis from testosterone to estradiol — in granulosa cell could be affected by gamma-radiation., U radu su procjenjivane strukture i endokrinološke promjene u folikulima jajnika štakorica i mišica izazvane gama-zračenjem. Štakorice su bile izložene zračenju od 3,2 Gy ili 8,0 Gy, a mišice od 2,9 Gy ili 7,2 Gy. Životinje su usmrćene dana 0, dana 4, odnosno dana 8 nakon ozračenja. Rezovi debljine 7 µm pripremljeni su za mikroskopiranje. Koncentracije progesterona, testosterona i estradiola u homogenatu jajnika određene su specifičnim radioimunoesejem. Gama-zračenje uzrokovalo je povećanje broja atretičnih folikula u obje skupine životinja usmrćenih 4 odnosno 8 dana nakon ozračivanja. Gama-zračenje također je smanjilo životni vijek granuloza stanica u skupinama usmrćenim 4. dan nakon ozračivanja. Utvrđeno povećanje omjera testosterona prema estradiolu u usporedbi s omjerom progesterona prema testosteronu upućuje na to da gama-zračenje utječe na aktivnost aromataze u steroidnoj biosintezi testosterona u estradiol u granuloza stanicama.

The pharmacokinetics and pharmacodynamics of testosterone enanthate and dihydrotestosteroneenanthate were compared in orchidectomized cynomolgus monkeys (Macaca fascicularis) and in intact GnRH agonist-suppressed rhesus monkeys (Macaca mulatta). Following a single im injection of 32.8 mg testosterone enanthate or 32.7 mg dihydrotestosteroneenanthate, i.e. 23.6 mg of pure steroid, in the orchidectomized cynomolgus monkeys, serum testosterone and dihydrotestosterone levels rose to 400 and 800% of baseline, respectively, within 24 h. Androgen levels remained in that range for 3-5 days followed by a continuous decline until baseline values were attained after 4-5 weeks. The areas under the testosterone- and dihydrotestosterone-curves did not differ significantly 2290±340 (dihydrotestosterone-enanthate) vs 2920±485 (testosteroneenanthate) suggesting that similar amounts of steroid had been released from the respective ester preparation. Mean half-life estimates of the terminal elimination phase were 4 and 7 days for testosterone-enanthate and dihydrotestosterone-enanthate, respectively. In a second experiment rhesus monkeys received, at 4-weekly intervals, sc implantation of a biodegradable polylactic:polyglycolide rod loaded with the GnRH agonist buserelin. The last injection was given during week 20. GnRH agonist treatment suppressed serum bioactive LH, testosterone and dihydrotestosterone levels, testicular size, sperm production, and seminal carnitine content. The ejaculatory response to electrostimulation and the masturbatory behaviour were abolished. Testosterone or dihydrotestosterone injections at the same doses as above were given in week 10, 14, 17 and 20 of GnRH agonist treatment. Serum testosterone and dihydrotestosterone levels were stimulated 9- and 4-fold, respectively. Mean half-life estimates for testosterone-enanthate and dihydrotestosterone were 5 and 7 days, respectively. Both ester preparations completely restored the ejaculatory response, ejaculate size, masturbatory behaviour, and seminal carnitine levels. In conclusion, androgen substitution with dihydrotestosterone-enanthate, in equivalent doses, is as effective as testosterone-enanthate in restoring reproductive functions in hypogonadal monkeys.

Purpose: Early detection and treatment of cancer is a primary focus of health care. Many serum markers are available for breast cancer, but are not good enough for screening. Cancer antigen CA 15-3 is the most widely used biomarker for breast cancer. However, CA 15-3 has low sensitivity and specificity. This study was performed to analyze the serum proteomic pattern in breast cancer patients by surface-enhanced laser desoption/ionization timeof-flight (SELDI-TOF). Methods: We screened for potential tumor biomarkers in 42 serum samples, including samples from a group of 23 breast cancer patients at different clinical stages [stage I (n=3), stage II (n=11), stage III (n=6), and stage IV (n=1)], and a control group of 19 healthy women. Diluted serum samples were applied to a C16 hydrophobic interaction chip (H4). Complex protein profiles of different groups were compared and analyzed using the Protein Chip software 2.1 (Ciphergen Biosystems). Results: There were 7 significant protein peaks in the breast cancer group and 5 in the control group. Scoring the expression of each peak, the mean score was 8.5 in the cancer group and 3.5 in the control. The results of the combination of each peak were highly sensitive (91.2%) and specific (94.7%). These proteomic patterns did not correlate with tumor stage and hormonal receptor, c-erb B2. Conclusion: In this preliminary report, we identified protein profiles that were differentiated in breast cancer patients. After proper validation, serum proteomic pattern analysis may ultimately be applied in screening breast cancer as a stand-alone or combined with current options. (Journal of Korean Breast Cancer Society 2004;7:22-26) ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ

A transdermal therapeutic system (TTS) for administration of testosterone was tested in healthy and hypogonadal men. To ensure adequate absorption, a testosterone-loaded film was applied to the scrotal skin. The TTS was designed to last for 22 h; over this time serum testosterone levels in normal men were moderately increased, with concentration curves almost parallel to basal levels. Seven hypogonadal patients also responded to TTS testosterone; serum testosterone levels were in the normal range during a 12-week treatment period. There were no side-effects. TTS testosterone offers a new approach to androgen substitution therapy.