• Advances in Traditional Medicine
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• v.6 no.2
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• pp.86-95
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• 2006

The ethanol extract of the Crotalaria juncea seeds, which showed promising antispermatogenic and antiandrogenic activities in albino mice, was taken up further for the isolation of the active fractions present in it. Two fractions that were obtained from thin layer chromatography were subjected for testing to know their antispermatogenic and antiandrogenic activities. After preliminary trials the fraction I showed maximum antifertility activity at the dose level of 200 mg/kg body weight when administered orally to the rats for 50 days. The fraction I was found to affect spermatogenesis as well as the endocrine functions of the testis as indicated by gravimetric, histopathological and biochemical changes. Further this fraction has caused degenerative changes in the seminiferous tubules and Leydig cells of the testis. The accessory reproductive organs like epididymis, seminal vesicles, vas deferens, prostrate, Cowper's gland and Levator Ani muscle showed significant malfunction. Cauda epididymal sperm count and sperm motility were reduced significantly. The treatment has also resulted in increase in the cholesterol level and alkaline phosphatase activity, and decrease in protein, glycogen, sialic acid contents and acid phosphatase activity in testis. It is noteworthy that RIA studies have shown significant reduction in serum FSH, LH and testosterone. Scanning electron microscopic observations revealed abnormalities in sperm structure.

• Journal of Embryo Transfer
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• v.28 no.2
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• pp.157-162
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• 2013

Methoxychlor (MXC) was developed to be a replacement for the banned pesticide DDT. HPTE [2,2-bis (p-hydroxyphenyl)-1,1,1-trichloroethane], which is an in vivo metabolite of MXC, has strong oestrogenic and anti-androgenic effects. MXC and HPTE are thought to produce potentially adverse effects by acting through oestrogen and androgen receptors. Of the two, HPTE binds to sex-steroid receptors with greater affinity, and it inhibits testosterone biosynthesis in Leydig cells by inhibiting cholesterol side-chain cleavage enzyme activity and cholesterol utilisation. In a previous study, MXC was shown to induce Leydig cell apoptosis by decreasing testosterone concentrations. I focused on the effects of MXC on male mice that resulted from interactions with sex-steroid hormone receptors. Sex-steroid hormones affect other organs including the kidney and liver. Accordingly, I hypothesised that MXC can act through sex-steroid receptors to produce adverse effects on the testis, kidney and liver, and I designed our experiments to confirm the different effects of MXC exposure on the male reproductive system, kidney and liver. In these experiments, I used pre-pubescent ICR mice; the puberty period in ICR mice is from postnatal day (PND) 45 to PND60. I treated the experimental group with 0, 100, 200, 400 mg MXC/kg b.w. delivered by an intra-peritoneal injection with sesame oil used as vehicle for 4 weeks. At the end of the experiment, the mice were sacrificed under anaesthesia. The testes and accessory reproductive organs were collected, weighed and prepared for histological investigation. I performed a chemiluminescence immune assay to observe the serum levels of testosterone, LH and FSH. Blood biochemical determination was also performed to check for other effects. There were no significant differences in our histological observations or relative organ weights. Serum testosterone levels were decreased in a dose-dependent manner; a greater dose resulted in the production of less testosterone. Compared to the control group, testosterone concentrations differed in the 200 and 400 mg/kg dosage groups. In conclusion, I observed markedly negative effects of MXC exposure on testosterone concentrations in pre-pubescent male mice. From our biochemical determinations, I observed some changes that indicate renal and hepatic failure. Together, these data suggest that MXC produces adverse effects on the reproductive system, kidney and liver.

• Journal of Animal Science and Technology
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• v.63 no.6
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• pp.1247-1264
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• 2021

Anabolic steroids are frequently used to increase the growth rate of meat-producing animals. Exposure to an anabolic-androgenic steroid, nandrolone decanoate (ND), is associated with expressional reduction of testicular steroidogenic enzymes. However, the effect of withdrawal of ND exposure on the expression of these testicular molecules has not been thoroughly explored. The current research investigated expression changes of testicular steroidogenic enzymes in rats at several recovery periods (2, 6, and 12 weeks) after the stop of ND treatment with different doses (2 and 10 mg/kg body weight) for 12 weeks. Body and testis weights were recorded, and transcript levels of molecules were determined by quantitative real-time polymerase chain reaction (PCR). The immunohistochemistry was used to examine the changes of immuno-intensities of molecules. At 6 and 12 weeks of the recovery period, the 10 mg/kg ND-treated rats were lighter than other experimental groups. The interstitial compartment vanished by ND treatment filled up as the recovery period became longer. The expression of steroidogenic acute regulatory protein was returned to the control level at 12 weeks of the recovery period. Expression levels of cytochrome P450 side-chain cleavage and 17a-hydroxylase were increased in 2 mg/kg ND-treated group at 6 weeks of the recovery period, and transcript levels of these molecules in 2 and 10 mg/kg ND-treated groups at 12 weeks of the recovery period were significantly lower than the control. Expression levels of 3β-hydroxysteroid dehydrogenase (HSD) type I and 17β-HSD type 3 in 2 mg/kg ND-treated group were comparable with those of control at 12 weeks of the recovery period, but not in 10 mg/kg ND-treated group. Expression of cytochrome P450 aromatase (Cyp19) was reverted to the control level at 2 weeks of the recovery period. Except for Cyp19, there was a visible increase of immuno-staining intensity of other testicular steroidogenic enzymes in the Leydig cells as the recovery period progressed. This research has demonstrated that the cease of ND administration could restore the expression of testicular steroidogenic enzymes close to the normal level. Nevertheless, a relatively long recovery period, compared to the ND-exposure period would be required to retrieve normal expression levels of testicular steroidogenic enzymes.

• Korean Journal of Environmental Biology
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• v.22 no.4
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• pp.550-558
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• 2004

The effects of postnatal exposure to octylphenol(OP) on the expressions of the steroidogenic enzymes and testosterone production were evaluated. Postnatal male mice (15-day-old) were injected with 2 or 20mg $kg^{-l}$ body weight (BW) of OP for 5 days and sacrificed on postnatal day 21. Testosterone concentration was measured by radioimmunoassay and the expressions of the testicular genes were determined by RT-PCR analyses. Significant reductions in the mean body and testis weight were observed in the OP treated animals. No marked alteration in the histological structure of the testis were observed, however, slight reduction in the seminiferous tubule diameter and the number of Leydig cells and several pyknotic cells could be identified in the 20 mg $kg^{-l}$ BW of the OP treated animals. Serum testosterone concentration was dramatically reduced and the mRNA expressions of the steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc) and $17\beta$-hydroxylase/Cl7-20 lyase $(P450_{17\alpha})$ were decreased. No significant changes of the gene expressions of the steroidogenic factor-l (SF-I) and estrogen and androgen receptor after the OP treatment showed that the decreased expressions of the steroidogenic enzymes in the present study did not correlate with these genes. Altogether, the present study demonstrates that postnatal treatment of OP inhibits steroidogenesis by decreasing the transcriptional expressions of the StAR and steroidogenic enzymes. The alteration in steroidogenesis may adversely affect the normal development of the testis and sper- matogenesis.

• Journal of Animal Reproduction and Biotechnology
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• v.36 no.4
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• pp.292-298
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• 2021

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 transcription-polymerase 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.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.54-54
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• 2001

Human chorionic gonadotropin (hCG) is a member of the glycoprotein hormone family which includes FSH, hCG, TSH. These hormone family is characterized by a heterodimeric structure composed a common $\alpha$-subunit noncovalently linked to a hormone specific $\beta$-subunit. The correct conformation of the heterodimer is also important for efficient secretion, hormone-specific post-translational modifications, receptor binding and signal transduciton. To determine $\alpha$ and $\beta$-subunits can be synthesized as a single polypeptide chain (tethered-hCG) and also display biological activity, the tethered-hCG molecule by fusing the carboxyl terminus of the hCG $\beta$-subunit to the amino terminus of the $\alpha$-subunit was constructed and transfected into chinese hamster ovary (CHO-K1) cells. We also constructed C-terminal deletion mutants (D9l, D89, D88, D87, D86, D84, D83) of single chain hCG to determine the biological function (secretion, LH-activity, receptor binding, cAMP production) of these mutants. Between six and eight stably transfected pools of cells expressing wild type and mutant hCGs were selected for neomycin resistant. The hCGs secreted by the stably transfected cells into serum-free media were collected and quantified by radioimmunoassay, as described in protocol (DPC(hCG IRMA). LH activity was in terms of testosterone production and aromatase activity in primary cultured rat Leydig cells. The tethered-wthCG was efficiently secreted and showed similar LH-like activity to the dimeric hCG. The D83hCG mutant was not detected in this assay. It is suggest that hCG C-terminal part is very important for hCG secretion. Now, we checking the LH-like activity of these mutant hCGs. These data indicate that the constructs of tethered molecule will be useful in the study of mutants that affect subunit association and/or secretion.

• Development and Reproduction
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• v.21 no.2
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• pp.111-120
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• 2017

Equine chorionic gonadotropin (eCG) is a unique molecule that elicits the response characteristics of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in other species. Previous studies from this laboratory had demonstrated that recombinant eCG (rec-eCG) from Chinese hamster ovary (CHO-K1) cells exhibited both FSH- and LH-like activity in rat granulosa and Leydig cells. In this study, we analyzed receptor internalization through rec-eCGs, wild type eCG ($eCG{\beta}/{\alpha}$) and mutant eCG ($eCG{\beta}/{\alpha}{\Delta}56$) with an N-linked oligosaccharide at $Asn^{56}$ of the ${\alpha}-subunit$. Both the rec-eCGs were obtained from CHO-K1 cells. The agonist activation of receptors was analyzed by measuring stimulation time and concentrations of rec-eCGs. Internalization values in the stably selected rat follicle-stimulating hormone receptor (rFSHR) and rat luteinizing/chorionic gonadotropin receptor (rLH/CGR) were highest at 50 min after stimulation with 10 ng of $rec-eCG{\beta}/{\alpha}$. The dose-dependent response was highest when 10 ng of $rec-eCG{\beta}/{\alpha}$ was used. The deglycosylated $eCG{\beta}/{\alpha}{\Delta}56$ mutant did not enhance the agonist-stimulated internalization. We concluded that the state of activation of rFSHR and rLH/CGR could be modulated through agonist-stimulated internalization. Our results suggested that the eLH/CGRs are mostly internalized within 60 min by agonist-stimulation by rec-eCG. We also suggested that the lack of responsiveness of the deglycosylated $eCG{\beta}/{\alpha}{\Delta}56$ was likely because the site of glycosylation played a pivotal role in agonist-stimulated internalization in cells expressing rFSHR and rLH/CGR.

• Korean Journal of Veterinary Research
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• v.40 no.2
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• pp.361-371
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• 2000

It has been recently reported that 2-bromopropane (2-BP) induces male reproductive toxicity in both human and experimental animals. However, delayed effects of 2-BP on male reproductive system have not been investigated in detail. The present study was conducted to investigate the testicular toxicity of 2-BP and to determine the recovery of normal spermatogenesis in Sprague-Dawley rats. Male rats aged 5 weeks were administered 1,000mg/kg 2-BP by gavage daily for 4 weeks and sacrificed sequentially at 1, 2, 3, 4 and 12 weeks after initiation of 2-BP treatment. Testicular toxicity was evaluated qualitatively by histopathological examinations and quantitatively by reproductive organ weights, spermatid head count, and repopulation index. In the 2-BP treated rats, the body weights was significantly suppressed and the weights of testes and epididymides were also decreased in a time-dependent manner. On histopathological examination, spermatogonia in stages I-VI and preleptotene and leptotene spermatocytes in stages VII-IX were strongly depleted at 1 week of dosing. Spermatogonia were depleted extensively in all spermatogenic stages at 2 weeks. Continuing with the evolution of spermatogenic cycle, zygotene spermatocytes, pachytene spermatocytes, and round spermatids were sequentially depleted at 2, 3, and 4 weeks of dosing due to the depletion of their precursor cells. Vacuolization of Sertoli cells and spermatid retention were also observed at all time points, suggesting that 2-BP induced Sertoli cell dysfunction. At 12 weeks, after 8 weeks recovery, most of the tubules appeared severely atrophic and were lined by Sertoli cells only. Leydig cell hyperplasia in the interstitial tissue was also found. In addition, dramatic reductions in the number of spermatid heads and repopulation index were observed, indicating that 2-BP-induced testicular injury is irreversible. These results indicate that 4 weeks repeated-dose of 1,000mg/kg 2-BP results in a progressive germ cell loss due to the depletion of spermatogonia followed by long-term testicular atrophy in SD rats.

• Journal of Life Science
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• v.28 no.10
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• pp.1147-1155
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• 2018

Agaricus blazei mycelial liquid culture extract (ABMLCE) promoted the production of testosterone (TS) in TM-3 mouse Leydig testis cells. Now, we report that ABMLCE containing eritadenine (EA) as a minor constituent (15.3 mg/100 g) reduced $5{\alpha}-reductase$ 2 ($5{\alpha}-R2$) enzyme activity and dihydrotestosterone (DHT) content which are key constituents for the benign prostatic hyperplasia (BPH) inductions. RWPE-1 prostate cells were grown in a Keratinocyte serum-free medium (K-SFM) containing ABMLCE (0~50 ppm), EA (0~10 ppm,), and finasteride (FS $10{\mu}M$: a positive control) in a 24-well plate for 24 hr. Supernatants collected from cell-cultured media were used for the assay of $5{\alpha}-R2$, superoxide dismutase (SOD), catalase (CAT) and cyclooxygenase-2 (COX-2) enzyme activities, and for TS, DHT, tumor necrosis $factor-{\alpha}$ ($TNF-{\alpha}$) and $interleukin-1{\beta}$ ($IL-1{\beta}$) contents by their assay kits. The $5{\alpha}-R2$ activity and DHT content were proportionally reduced (p<0.05) to concentrations of ABMLCE. The SOD and CAT enzyme activities were significantly (p<0.05) elevated concomitant with ABMLCE concentrations, while COX-2, $TNF-{\alpha}$ and $IL-1{\beta}$ showed reverse results (p<0.05). Similarly, the effects of EA were similar to those of ABMLCE. Efficacies of ABMLCE 50 ppm and EA 10 ppm in $5{\alpha}-R2$ and DHT reduction were similar to those of $10{\mu}M$ FS. These results suggest that ABMLCE and EA reduced $5{\alpha}-R2$ and DHT through their anti-inflammatory and anti-oxidative actions. This implies that ABMLCE containing EA could be a beneficial material in the cure of BPH in humans.

• Development and Reproduction
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• v.10 no.3
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• pp.203-209
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• 2006

Ethane 1,2-Dimethane sulfonate(EDS), a toxin which specifically kills Leydig cells(LC), has been widely used to prepare the reversible testosterone(T) depletion rat model. Previous studies including our own clearly demonstrated that the dramatic weight loss of the T-dependent accessory sex organs such as epididymis and seminal vesicle in this 'LC knock-out' rats. These weight loss could be derived from massive and abrupt death of the cells via apoptotic process. The present study was performed to test the effect of EDS administration on the expression of some apoptotic genes in the rat epididymis. Adult male Sprague-Dawley rats($300{\sim}350$ g B.W.) were injected with single dose of EDS(75 mg/kg, i.p.) and sacrificed on Weeks 0, 1, 2, 3, 4, 5, 6 and 7. Tissue weights and the numbers of the epididymal sperm were measured. The transcriptional activities of the bcl-2, bax, Fas and Fas ligand(Fas-L) were evaluated by semi-quantitative RT-PCR. As expected, the weights and the sperm counts of epididymis declined progressively after the EDS treatment during Week 1 and 2. These decrements were discontinued with a gradual return towards normal during Weeks $5{\sim}7$, although the maximal recoveries of the epididymal weights(71%) and sperm count(38%) were subnormal on Week 7. The initial level of bcl-2 transcripts persisted to Week 6 then elevated significantly on Week 7. The level of bax transcripts significantly decreased on Week 6, and no remarkable change was found in the rest of the experimental period. The transcripts for the Fas in epididymis elevated during Weeks $1{\sim}2$, returned to normal on Week 3, and the level persisted to the Week 7. Similarly, the level of Fas-L transcripts elevated during Weeks $1{\sim}3$ and returned to normal after Week 4. Our results demonstrated the transient T depletion by EDS administration could induce the changes in expression of the apoptotic genes in rat epididymis. The activation of Fas and Fas-L in the epididymis of EDS-treated rats might be responsible for the initial apototic process and consequently the tissue damage and the sperm loss. Future studies will attempt to determine the precise molecular mechanism(s) of apoptosis in the rat epididymis.