• Clinical and Experimental Reproductive Medicine
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• v.40 no.4
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• pp.155-162
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• 2013

Objective: Stress is known to be an inhibitor of the reproductive hypothalamic-pituitary-gonadal (HPG) axis. However, the neural and molecular connections between stress and reproduction are not yet understood. It is well established that in both humans and rodents, kisspeptin (encoded by the kiss1 gene) is a strong stimulator of the HPG axis. In the present study we hypothesized that endocannabinoids, an important neuromodulatory system in the brain, can act on the HPG axis at the level of kiss1 expression to inhibit reproductive function under stress. Methods: Adult male Wistar rats were unilaterally implanted with an intracerebroventricular cannula. Afterwards, the animals were exposed to immobilization stress, with or without the presence of the cannabinoid CB1 receptor antagonist AM251 (1 ${\mu}g/rat$). Blood samples were collected through a retro-orbital plexus puncture before and after stress. Five hours after the stress, brain tissue was collected for reverse transcriptase-quantitative polymerase chain reaction measurements of kiss1 mRNA. Results: Immobilization stress (1 hour) resulted in a decrease in the serum luteinizing hormone concentration. Additionally, kiss1 gene expression was decreased in key hypothalamic nuclei that regulate gonadotrophin secretion, the medial preoptic area (mPOA), and to some extent the arcuate nucleus (ARC). A single central administration of AM251 was effective in blocking these inhibitory responses. Conclusion: These findings suggest that endocannabinoids mediate, at least in part, immobilization stress-induced inhibition of the reproductive system. Our data suggest that the connection between immobilization stress and the HPG axis is kiss1 expression in the mPOA rather than the ARC.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.2
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• pp.174-183
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• 2001

During the rapid phase of gonadal development of the freshwater teleost, the catfish (Silurus asotus), the influence of hCG upon the inducement of final oocyte maturation and spawning was investigated electrophoretically and ultrastructurally. The electrophoretic patterns obtained were different in the presence and absence of some of the major or minor zones, because of the hormone level in catfish. The vitellogenin of hormone-treated fish was stained more intensively than that of sham-treated fish. These proteins showed some minor or main bands of egg extracts which migrated at positions corresponding to molecular weights of approximately 90,000. However, the thickness of electrophoretic band in molecular weight for hCG-treated fish was slightly lower than that for saline control. It seemed the plasma protein with molecular weight of approximately 45,000 in hCG-treated fish disappeared. In contrast to the control fish, the ovaries in the catfish treated with hCG shows a marked ultrastructural change under the electron microscope. No dilated profiles were seen in the granulosa cells of the mature oocyte before ovulation. After germinal vesicle breakdown (GVBD), the zona radiata interna (ZRI) becomes more compact, and there is a loss of all the processes from the pore canals. There is a wide space between the vitelline membrane and zona radiata. Also, during final maturation, the microvillar processes from the oocyte are seen no longer to penetrate deeply into the extracellular spaces of the overlying granulosa cells, and the reticulate patterns of the zona radiata interna becomes occluded, giving the zona radiata a more solid appearance. It has been possible to initiate 100% oocyte maturation in yolk granules and follicles in vivo by treatment with hCG and a high water temperature ($27^{\circ}C$). In hCG-treated fish, the percentages of successful artificial fertilization and hatching were maximal at 15 h after a single injection. It seems clear that a long acting preparation containing hCG can be successfully used in prespawning fish to advance the final events of gonadal maturation and initiate spawning. Further studies are necessary to evaluate the potential of hCG to either stimulate or inhibit the reproductive development of fish at other stages of the seasonal reproductive cycle.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.38 no.5
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• pp.309-315
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• 2005

Changes of sex steroid hormones in the plasma of yellowfin goby, Acanthogobius flavimanus were investigated in relation to the gonadosomatic index (GSI), the hepatosomatic index (HSI) and gonadal development. The GSI in females rose rapidly in November and remained high from December to May $(7.26\pm0.89­6.62\pm0.02)$. The Male's GSI also increased gradually from November and was highest in May $(0.16\pm0.08)$. The HSI in both sexes was in reverse correlation with the GSI, and the HSI was low during the spawning season (February-May). In females, the $estradiol-17{\beta}\;(E_2)$ level increased during vitellogenesis (November and December) and reached its maximum $(8.13\pm2.87 ng/mL)$ at the maturing period, in January. $17{\alpha},\;20{\beta}$-dihydroxy-4-pregnen-3-one$(17{\alpha}20{\beta}OHP)$ gradually increased from October $(0.063{\pm}0.02ng/mL)$ to March $(0.16{\pm}0.02ng/mL)$ and increased rapidly in May. The level of testosterone (T) showed a similar tendency of $E_2$. In males, T increased gradually during spermatogenesis from September to December $(0.14{\pm}0.06­0.26{\pm}0.10ng/mL)$ and peaked in January $(0.36{\pm}0.29 ng/mL)$ when the spermatozoa filled the testis. 11-KT also rose markedly in January and then decreased. On the other hand, $17{\alpha}29{\beta}OHP$ in males did not show any clear tendencies.

• Development and Reproduction
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• v.5 no.1
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• pp.1-8
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• 2001

Golden hamsters show the reproductive activity that is determined by the photoperiod (length of light per day). Photoperiod is an environmental factor that is predictable through an entire year. The hamsters are sexually active in summer during which day length exceeds night time. The critical length is at least 12.5 hours of light in a day where reproductive function is maintained. The information of photoperiod is mediated by the pineal gland because removal of pineal gland blocks the influence of photoperiod on reproductive activity. The hamsters without pineal gland maintain sexual activity and promote it in a situation that suppresses gonadal activity. The pineal gland secretes melatonin that reflects the photoperiod. The appropriate administrations of melatonin into both pineal intact and pinealectomized hamsters lead to a gonadal reression. The results suggest that melatonin constitutes a part of control mechanism whereby environmental information is transduced to neuroendocrine signal respensible for the functional integrity of the reproductive system. Despite of the intense studies, the action site of melatonin is on the whole unknown. It is mainly due to the lack of acute efffct of melatonin on the secretion of reproductive hormones. However, sexually regressed animals display the low levelsof gonadotropins and the augmentation of the hypothalamic gonadotropin-releasing hormone (GnRH) content, implying that the antigonadotropic effects either by photoperiod and/or by the treatment of melatonin are mediated by the GnRH neuronal system. The action mechanism by which melatonin exerts its effect on GnRH neuron needs to be investigated. Recent cloning of melatonin receptor will contribute to examine various and putative potencies of melatonin via its anatomical identification and the action mechanism of melatonin on target tissues at the molecular level.

• The Korean Journal of Zoology
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• v.39 no.4
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• pp.337-351
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• 1996

Melatonin Is a multifunctional hormone secreted from the pineal gland in the middle of cerebrum and cerebellum. Its synthesis and release reflect photopedod;Photopedod is a yearly predictable ambient factor that most animals utilize as an environmental cue for maximum survival. Hamsters maintaln reproductive activity in summer during which day length exceeds night time. Upon the advent of autumnal equinox they undergo gonadal regression. The photoperiodic effects are prevented by removal of the pineal gland and restored by the timed repiacument of melatonin. The results suggest that melatonin constitutes part of control mechanism whereby environmental information is transduced to neuroendocrine signal responsIble for the functional integrity of the reproductive system. From the studies for the action site of melatonin following the treatment of photopedod or melatonin in the lesion of a spedflc portion of hypothalamus, suprachiasmatic nuclei and pars tuberalis are shown to be a consensus site for melatonIn. The action of melatonin. In the regulation of reproduction is largely unknown. It is mainly due to the lack of acute effect of melatonin on gonadotropin secretion. However, reduction of the gonadotropln release and augmentation of the hypothalamic gonadotropin-releasing hormone (GnRH) content by long-term treatment of melatonln Indicate that constant presence of melatonln may partidpate in the regulation of sexual activity via the GnRH neuronal system. The action mechanism by which melatonin exerts Its effect on GnRH neuron needs to be eluddated. The inability of opiold analogues to affect the reproductive hormones in sexually regressed animals by inhibftory photopedod and melatonin suggests that the opioldergic neuron may be a prime intervening mediator. Recent cloning of melatonin receptor will contribute to investigate its anatomical Identification and the action mechanism of melatonin on target tissues at the molecular level.

• Animal Bioscience
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• v.35 no.12
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• pp.1850-1859
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• 2022

Objective: The quantitative reverse transcription polymerase chain reaction (qPCR) is the most accurate and reliable technique for analysis of gene expression. Endogenous reference genes (RGs) have been used to normalize qPCR data, although their expression may vary in different tissues and experimental conditions. Verification of the stability of RGs in selected samples is a prerequisite for reliable results. Therefore, we attempted to identify the most stable RGs in the hypothalamic-pituitary-gonadal (HPG) axis in sows. Methods: The cycle threshold values of nine commonly used RGs (18S, HPRT1, GAPDH, RPL4, PPIA, B2M, YWHAZ, ACTB, and SDHA) from HPG axis-related tissues in the domestic sows in the different stages of estrus cycle were analyzed using two RG-finding programs, geNorm and Normfinder, to rank the stability of the pool of RGs. In addition, the effect of the most and least stable RGs was examined by normalization of the target gene, gonadotropin-releasing hormone (GnRH), in the hypothalamus. Results: PPIA, HPRT1, and YWHAZ were the most stable RGs in the HPG axis-related tissues in sows regardless of the stages of estrus cycle. In contrast, traditional RGs, including 18S and ACTB, were found to be the least stable under these experimental conditions. In particular, in the normalization of GnRH expression in the hypothalamus against several stable RGs, PPIA, HPRT1, and YWHAZ, could generate significant (p<0.05) elevation of GnRH in the preovulatory phase compared to the luteal phase, but the traditional RGs with the least stability (18S and ACTB) did not show a significant difference between groups. Conclusion: These results indicate the importance of verifying RG stability prior to commencing research and may contribute to experimental design in the field of animal reproductive physiology as reference data.

• Development and Reproduction
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• v.1 no.2
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• pp.189-202
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• 1997

The hypothalamic peptide GnRH plays a central role in the regulation of the mammalian reproductive axis. Recent studies suggested that GnRH stimulates or inhibits the ovarian steroidogenesis and gametogenesis directly. Our previous report indicated that GnRH gene is expressed in adult rat ovary as well as in hypothalamus and that the expressed GnRH may induce the follicular atresia and apoptosis of ovarian granulosa cells in rat. Therfore, we studied whether GnRH gene is expressed in the mouse fetal ovary, when the germ cells are degenerating by apoptosis during gonad diffeerentiation. Mouse fetal gonads were obtained on the 12, 15,18 and 20th day of gestation from the mother mice superovulated (10 IU PMSG and 10 IU hCG) and mated. The morphological changes of fetal ovaries were examined histochemically by hematoxylin-eosin staining. The fetal sex was confirmed by PCR methods for sexing. RT-PCR methods were used to examine the expression of GnRH gene and the sex steroid hormones were determined by conventional radioimmunoassays. The levels of estradiol (E) and progesterone (P) were increaseduntil 18th day of gestation and then E was decreased just before parturition. The morphological changes of fetal gonadal tissue sections showed the ovarian development and coincided with the result of PCR analysis for sexing using ovary- or testis- specific oligonucleotide primers. Immunoreactive GnRH in placenta was decreased gradually until the end of gestation but fetal brain and ovarian GnRH were increased. The level of GnRH gene expression was increased during fetal ovarian development from 12 till 18th day and decreased suddenly on 20th day just before birth. From these results, it is suggested that ovarian GnRh may play a regulatory role on the germ cell differentiation of fetal ovary.

• Development and Reproduction
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• v.7 no.1
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• pp.35-40
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• 2003

Gonadotropin releasing hormone(GnRH), which is secreted from the hypothalamus, has a pivotal role in the reproduction of mammals. Golden hamsters are seasonal breeding mammal and their sexual activity is determined by photoperiod(length of light per day). Long photoperiod(LP, $\leq$ 12.5 hours of light) maintains the reproductive activity and short photoperiod(SP, $\leq$ 12 hours of light) suppresses it. In order to investigate in detail, the sexual activity was individually examined in SP-housed male golden hamsters received a vector at three different concentrations which contains rat GnRH cDNA. The gonadal regression was significantly(P<0.05) accelerated by the highest concentrations of the vector at 8 and 10 weeks after the treatment in comparison to the other groups. In the light of pulsatile release of GnRH in maintaining reproductive activity, the vector containing GnRH cDNA might secrete the GnRH in a constant high level. These results suggest that the GnRH-containing vector might desensitize the anterior pituitary, leading to acceleration or testicular regression.

• Clinical and Experimental Pediatrics
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• v.54 no.5
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• pp.224-227
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• 2011

A sclerosing stromal tumor of the ovary is an extremely rare benign tumor; it usually is found during the second and third decades of life. Patients present with pelvic pain or a palpable abdominal mass. Hormonal effects such as masculinization are uncommon. Here, an 11-year old premenarchal girl presented with deepening of the voice. In addition, clitoromegaly and hirsutism with a male suprapubic hair pattern were observed. The laboratory findings showed that the testosterone level was elevated to 3.67 ng/ml, andostenedione to above 10 ng/ml, dehydroepiandrosterone-sulfate to 346 ${\mu}g$/dl and 17-hydroxy progesterone (17-OHP) to 11.28 ng/ml. The chromosome evaluation revealed a 46,XX female karyotype. An adrenocorticotropic hormone stimulation test was performed. The 17-OHP to cortisol ratio in 30 minutes was 0.045, which suggested a heterozygote for the 21-hydroxylase deficiency. However, the CYP21A2 gene encoding steroid 21-hydroxylase showed normal. The pelvic ultrasound showed a heterogeneous mass consisting of predominantly solid tissue in the pelvic cavity. The pelvic magnetic resonance imaging revealed an $8.9{\times}6.2{\times}6.6$ cm mass of the left ovary. A left oophrectomy was performed and microscopic examination confirmed a sclerosing stromal tumor. Immunohistochemical studies showed that the tumor was positive for smooth muscle actin and vimentin, but negative for S-100 protein and cytokeratin. Following surgery, the hormone levels returned to the normal range and the hirsutism resolved.

• Advances in Traditional Medicine
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• v.10 no.2
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• pp.103-110
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• 2010

The seed powder of Abrus precatorius L. has traditionally been used as oral contraceptive agent by the women in some rural areas in Bangladesh. The present study aimed to examine the antifertility activity of A. precatorius seed extracts in experimental female rats. Finely ground seeds were extracted with aqueous acetone followed by successive partitioning with n-hexane, ethyl acetate (EtOAc), methanol (MeOH) and water. Water suspended crude seed powder, organic fractions of acetone extract and a standard contraceptive drug ($Nordette^{(R)}28$) were separately administered orally to the female rats for 30 days. n-Hexane, EtOAc and MeOH solubles at the doses of 2, 4 and 6 mg/rat/day, respectively and crude seed powder at 100 mg/rat/day exhibited 100% antifertility activity with lowest levels of serum luteinizing hormone (LH), follicle stimulating hormone (FSH) and $17{\beta}$-estradiol. Histological study of ovary and uterus of these rats exhibited reduced number of developing follicles and increased number of atretic follicles in the ovary, and fewer uterine glands with shrunken morphology, reduced endometrial height, poor vascularity and compact stroma in uterus. However, the activities of serum glutamate oxaloacetate transaminase and serum glutamate pyruvate transaminase and the body weight of the rats remained almost unaffected in all the seed extract treated rats compared to control. These results suggest that A. precatorius seed extracts reduced the levels of serum FSH, LH and $17{\beta}$-estradiol probably by affecting hypothalamic-pituitary-gonadal axis. The reduced levels of these hormones might have affected the oestrous cycle, follicular development, and subsequently the establishment of pregnancy in treated rats.