• Asian-Australasian Journal of Animal Sciences
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• v.24 no.4
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• pp.463-470
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• 2011

Gonadotropin-releasing hormone receptor (GnRHR) gene is expressed at the anterior pituitary gland and plays a key role in gonad development. This study aimed to investigate molecular genetic characteristics of the GnRHR gene and elucidate the effects of single nucleotide polymorphisms (SNPs) of GnRHR gene on sex steroid level in Japanese flounder (Paralichthys olivaceus). We used polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) and sequencing of the GnRHR gene in 75 individuals. We identified three SNPs in the GnRHR gene: P1 locus (C759A and C830T) in the coding region of exon2 which were both linked together and P2 locus (G984T) in the coding region of exon3, which added a new transcript factor (ADR1) and a new methylation site (CG). Only C830T of P1 leads to amino acid changes Thr266Ile. Statistical analysis showed that P1 was significantly associated with $17{\beta}$-estradiol ($E_2$) level (p<0.01) and gonadosomatic index (GSI) (p<0.05). Individuals with genotype BB of P1 had significantly higher serum $E_2$ levels (p<0.01) and GSI (p<0.05) than those of genotype AA or AB. Another SNP, P2, synonymous mutation, was significantly associated with GSI (p<0.05). Individuals with genotype AB of P2 had significantly higher GSI (p<0.05) than that of genotype AA. In addition, there was a significant association between one diplotype based on three SNPs and reproductive traits. The genetic effects for both serum $E_2$ level and GSI of diplotype D4 were super diplotypes (p<0.05). These results suggest that the SNPs in Japanese Flounder GnRHR are associated with $E_2$ level and GSI.

• Applied Microscopy
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• v.41 no.1
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• pp.37-45
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• 2011

Known as a female hormone, estrogen, has an effect on the male reproductive organs. The estrogen has to combine with the estrogen receptor to communicate a signal. Propyl pyrazole triol (PPT) is an estrogen receptor alpha selective agonist with a 410-, or 1,000-fold relative binding affinity for estrogen receptor alpha versus estrogen receptor beta. In this study, adult male mice were treated weekly with subcutaneously injections of PPT (0.01 mg, 0.1 mg, 1mg and 4 mg) suspended in castor oil (as control) for 8 weeks and observed histologically changes in testis, efferent ductule and epididymis. In the high concentrations of PPT 4 mg treatment group, a remarkable reduction was observed in the weight of the body, testis and epididymis. Microscopic examination revealed a reduction in seminiferous tubular diameter of the testis, and epithelial cell height of the epididymis in treated group during the experiment. In addition, as the diameter of the efferent ductule increased gradually, the height of epithelial cells was decreased. PPT 4 mg treatment group caused inhibition of spermatogenesis due to atrophied germinal epithelium in the testis, and decrease of adipocyte size attached to the epididymis. Sperm was not observed in the caudal epididymis of PPT 4 mg treated group. In conclusion, the injection of high concentrations of PPT into adult male mice induced physiological changes, such as an inhibition of spermatogenesis, and also histological changes within the reproductive organs.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.3
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• pp.283-292
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• 1996

Objective: To determine the cutoff value of clomiphene citrate challenge test(CCCT) that can predict the normal and abnormal(diminished) ovarian response group and to assess the usefulness of CCCT as a predictor of ovarian reserve. Materials and Methods: From March 1994 to Februry 1996, CCCT was performed to 129 infertile patients and among them, 20 patients whose basal FSH on the third day of menstrual cycle was more than 20 mIU/ml were excluded. At the same time, the same CCCT was performed to the fifteen healthy volunteers with proven fertility to determine the cutoff value of CCCT. Results; 1) A FSH value higher than 23.4 mIU/ml, measured on the 10th day of menstrual cycle, was defined as a abnormal ovarian response. The cutoff value of 23.4 mIU/ml is more than 2 standard deviations(SD) above the mean value of 15 healthy women after CCCT. 2) The abnormal CCCT group, the subpopulation with a FSH value of 23.4 mIU/ml or more, was 7.3%(8/109) and their mean age was higher than the normal CCCT group($36.5{\pm}4.5$ vs. $32.9{\pm}4.8$, P = 0.059). And the percentage of the patients older than 35 years of the abnormal CCCT group was significantly higher than that of the normal CCCT group(62.5% vs. 38.6%, p <0.05). 3) There was no correlation between the hormone values of the third day and the 10th day of menstrual cycle before and after CCCT except between FSH of the third day and the 10th day. Conclusion: The CCCT is a good method to predict the individual ovarian response to COH for ART, especially in the patients who has no other abnormal findings that predict poor prognosis. And it is neccessary to determine the cutoff value of CCCT by the large numbers of randomized study, and combining the previously proven prognostic factors, it can be applicated in many individual centers for evaluate the ovarian response before ART program.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.35 no.6
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• pp.614-620
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• 2002

The sea bass, Lateoiabrax japonicus is a highly valued fish in aquaculture in South Korea. For establishment of seedling production of sea bass,1 japonicus, we examined change of gonadal development and plasma steroid levels of sea bass reared in indoor tank. Male matured unsimultaneously faster than females and spawning of females took place between the end of January and March. After the spawning period, and until the following January, all the females were in preyitello genesis and in some males, spermatogenetic activity restarted gradually. In October, under reducing photoperiod, cortical alveoli appeared in growing oocyte and the development of spermatogenesis greatly increased. Between October and february, vitellogenesis and spermatogenesis occurred respectively in female and male and gonadosomatic index increased from 4.31 to $24.07\%$ in female and upper 6o/o in male. Also, two sex hormones were analyzed during the course of a reproductive cycle in the sea bass: plasma levels of the gonadal steroid testosterone (T) and estradiol-l7$\beta$ (E_{2}). Variation of the plasma concentrations of T and E, appeared to depend on gonad stages. Plasma T and E, levels were high from November to January, suggesting that an sufficient gonadal stimulation by both hormones may undergoing a processes for the formation of sperm and oocyte.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.1
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• pp.51-59
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• 1996

Controlled Ovarian hyperstimulation(COH) is generally used to obtain synchronous high quality oocytes in in vitro fertilization-embryo transfer(IVF-ET). Many investigators have studied the relationship between serum hormone levels and outcomes of IVF-ET because there is no accurate estimation method of oocyte quality. Early premature luteinization of follicles before oocyte retrieval is the most troublesome problem in COH for IVF-ET. Gonadotropin-releasing hormone agonists(GnRH-a) are used as adjuncts with gonadotropins for COH in patients undergoing in IVF. The possible benefits of GnRH-a pretreatment include improving oocyte quality, allowing a more synchronous cohort of follicles to be recruited, and preventing premature lueinization hormone surges. In COH of IVF cycles, we investigated whether an elevated progesterone(P4) level on the day of human chorionic gonadotropin(hCG) administration indicates premature luteinization and is associated with a lower fertilization rate. Many investigators have studied that the lower fertilization rates seen in patients with elevated P4 levels might result from an adverse effect of P4 on the oocytes. We hypothesizes that serum P4 levels around the day of hCG may be helpful prediction of out come in IVF-ET cycles. Success rates after COH of IVF-ET cycles are dependent upon many variable factors. Follicular factors including the number of follicles, follicular diameters and especially serum estradiol(E2) levels as an indirect measurement of follicular function and guality have been thought to influence the outcomes of IVF-ET. To assess whether serum P4 and E2 levels affect the fertilization and pregnancy rate, we reviewed the stimulation cycles of 113 patients (119 cycles) undergoing IVF-ET with short protocol with GnRH-a, from March 1993 to August 1994 retrospectively. The serum P4 and E2 levels were compared on the day of hCG in the pregnant group, 45 patients(47 cycles) and in the non-pregnant group, 68 patients (72 cycles) respectively. The serum E2 level in non-pregnant group was $1367{\pm}875.8$ pg/ml which was significantly lower than that of pregnant group, $1643{\pm}987.9$ pg/ml( p< 0.01 ). And the serum P4 level in non-pregnant group was $2.1{\pm}1.4$ ng/ml which was significantly higher than that of pregnant group, $1.0{\pm}0.7$ ng/ml( p< 0.001 ). The fertilization rate was $61.3{\pm}21.3%$ in pregnant group which was higher than that of non-pregnant group, $41.1{\pm}20.2%$ (p< 0.01). We suggest that the serum levels of P4 and E2 on the day of hCG administration are additional parameters that predict the outcomes of IVF-ET cycles.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.4
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• pp.487-499
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• 2016

The aim of the present study was to examine the effects of testosterone (T) and estradiol-$17{\beta}$ ($E_2$) on the production of progesterone ($P_4$) by granulosa cells, and of the $E_2$ on the production of $P_4$ and T by theca internal cells. In the first experiment, granulosa cells isolated from the largest ($F_1$) and third largest ($F_3$) preovulatory follicle were incubated for 4 h in short-term culture system, $P_4$ production by granulosa cells of both $F_1$ and $F_3$ was increased in a dose-dependent manner by ovine luteinizing hormone (oLH), but not T or $E_2$. In the second experiment, $F_1$ and $F_3$ granulosa cells cultured for 48 h in the developed monolayer culture system were recultured for an additional 48 h with increasing doses of various physiological active substances existing in the ovary, including T and $E_2$. Basal $P_4$ production for 48 h during 48 to 96 h of the cultured was about nine fold greater by $F_1$ granulosa cells than by $F_3$ granulosa cells. In substances examined oLH, chicken vasoactive intestinal polypeptide (cVIP) and T, but not $E_2$, stimulated in a dose-dependent manner $P_4$ production in both $F_1$ and $F_3$ granulosa cells. In addition, when the time course of $P_4$ production by $F_1$ granulosa cells in response to oLH, cVIP, T and $E_2$ was examined for 48 h during 48 to 96 h of culture, although $E_2$ had no effect on $P_4$ production by granulosa cells of $F_1$ during the period from 48 to 96 h of culture, $P_4$ production with oLH was found to be increased at 4 h of the culture, with a maximal 9.14 fold level at 6 h. By contrast, $P_4$ production with cVIP and T increased significantly (p<0.05) from 8 and 12 h of the culture, respectively, with maximal 6.50 fold response at 12 h and 6, 48 fold responses at 36 h. Furthermore, when $F_1$ granulosa cells were precultured with $E_2$ for various times before 4 h culture with oLH at 96 h of culture, the increase in $P_4$ production in response to oLH with a dose-related manner was only found at a pretreatment time of more than 12 h. In the third experiment, theca internal cells of $F_1$, $F_2$ and the largest third to fifth preovulatory follicles ($F_{3-5}$) were incubated for 4 h in short-term culture system with increasing doses of $E_2$. The production of $P_4$ and T by theca internal cells were increased with the addition of $E_2$ of $10^{-6}M$. These increases were greater in smaller follicles. These results indicate that, in granulosa cells of the hen, T may have a direct stimulatory action in the long term on $P_4$ production, and on $E_2$ in long-term action which may enhance the sensitivity to LH for $P_4$ production, and thus, in theca internal cells, $E_2$ in short term action may stimulate the production of $P_4$ and T.

• Korean Journal of Animal Reproduction
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• v.9 no.1
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• pp.9-30
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• 1985

The study was carried out to find out the changes of the sex hormone levels in the milk of Holstein cows during the reproductive stages such as the estrous cycle, pregnancy and periparturient period. The FSH, LH, estradiol-17$\beta$ and progesterone from the milk samples were assayed by radioimmunoassay methods. The results of this study were summarized as follows: 1. The levels of progesterone and estradiol-17$\beta$ were similar among inter-quarters, but they were higher in after milking than before milking times, with no statistical significance. 2. The milk progesterone levels during the estrous cycles reached a peak mean level of 3.55$\pm$0.26ng/$m\ell$ at 15 days after estrus and they did not show any differences among the length of estrous cycles. The estradiol-17$\beta$ levels during the estrous cycles showed a peak level of 36.40$\pm$2.38pg/$m\ell$ at estrus, and decreased(17.20$\pm$0.46 pg/$m\ell$ to 18.65$\pm$1.26pg/$m\ell$) at luteal phase. 3. The FSH levels during the estrous cycles ranged from 2.25$\pm$0.23mIU/$m\ell$ to 4.35$\pm$0.24mIU/$m\ell$ showing significant changes. The LH levels during the estrous cycles gradually increased and remained a peak level of 10.90$\pm$0.36mIU/$m\ell$ from 20 to 25 days after estrus. 4. The progesterone levels during the pregnancy were decreased from 30 to 60 days after artificial insemination, and therafter continuously increased until 240 days. The estradiol-17$\beta$ levels during the pregnancy were 24.56$\pm$1.19pg/$m\ell$ at day 30 after artificial inseminaton, and increased rapidly until 180 days. The levles were agagin decreased by 26.17$\pm$3.03pg/$m\ell$ until 210 days and markedly increased by 68.00$\pm$8.70pg/$m\ell$ until 240 days. 5. The prolactin levels during the pregnancy were 31.27$\pm$2.31ng/$m\ell$ and 42.60$\pm$2.37ng/$m\ell$ at day 150 and 240 after artificial insemination respectively. The LH levels during the pregnancy reached a peak of 27.47$\pm$7.90mIU/$m\ell$ at day 30 after artificial insemination, and thereafter gradually decreased. 6. The progesterone levels during the periparturient period reached a peak of 4.61$\pm$0.34ng/$m\ell$ at day 3 prepartum, and thereafter gradually decreased, and showed 2.05$\pm$0.60ng/$m\ell$ at day 7 postpartum. The estradiol-17$\beta$ levels during the periparturient period showed high level from 207.23$\pm$6.04pg/$m\ell$ at day 1 prepartum to 239.90$\pm$13.90pg/$m\ell$ at day 2 prepartum, and thereafter began to decline and reached 51.87$\pm$1.72pg/$m\ell$ at by 7 postpartum. 7. The prolactin levels during the periparturient period showed relatively higher level at the time of parturition. The LH levels during the periparturient period rnage from 6.32$\pm$0.32mIU/$m\ell$ to 13.90$\pm$1.37mIU/$m\ell$ showing significant changes. 8. The progesterone levels(4.6$\pm$0.8ng/$m\ell$) of the pregnant cows were significantly higher than those (1.84$\pm$1.4ng/$m\ell$) of nonpregnant cows. The cows of artificial insemination from 61 to 90 days after parturition showed higher progesterone levels. 9. During 20 to 25 days after artificial insemination, the accuracy of pregnancy diagnosis from milk progesterone levels were 94.4% for nonpregnant cows(<2.3ng/$m\ell$), and 75.0% for pregnant cows( 3.2ng/$m\ell$). The average overall accuracy of pregnancy prediction for nonpregnant and pregnant cows 83.3% 10. The results obtained this study suggest that the understanding of the endocrinological mechanisms by means of milk hormone analysis during the estrous cycle, pregnancy and parturition would give the basic information needed for increasing efficiency of reproduction. This study would not only provide an accurate method of the early pregnancy diagnosis by milk progesterone levels but also contribute to the research of providing the method of detecting of FSH levels in milk, which was difficult in blood serum.

• Journal of Veterinary Clinics
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• v.17 no.1
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• pp.225-233
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• 2000

The aim in this study was to obtain the basic data for estimation of optimal mating time and ovulation time by finding out the relationship between vaginal cytology and reproductive hormone profiles during the estrous cycle in 36 Korea Jin-do bitches. A characteristic features of vaginal cytology during the estorous cycle were the high proportion of superficial cell, anuclear cell and erythrocyte in proestrus, superficial cell, anuclear cell and erythroucyte in estrus, parabasal cell, small and large intermediate cell and leukocytes in diestrus, and parabasal cell and small intermediate cell in anestrus, respectively. Cornification index(CI) was the high proportion in proestrus and estrus, then it was decreaed in diestrus and anestrus. It indicated that the CI was significantly high proportion in proestrus and estrus in comparison with the other phases. Plasma progesterone concentration was below 1.0ng/ml at the first day of vulval bleeding in pregnant and non-pregnat bitches, and then it was increaed slowly. When Day 0 was timed from the day that plasma progesterone concentration was first increased above 4.0ng/ml, plasma progesterone concentrations at Day 0 in pregnant and non-pregnant above 4.0 ng/ml, plasma progesterone concentrations at Day 0 in pregnant and non-pregnant bitches were $5.2{\pm}0.3ng/ml and 5.7{\pm}0.5ng/ml$$46.5{\pm}3.3 ng/ml$ in pregnant bitches and at Day 20 with $39.8{\pm}0.1ng/ml$ in nonpregnant bitches. It indicated that plasma progesterone concentration was higher in pregnant bitches than in non-pregnant bitches. Hereafter plasma progesterone concentration was decreased steadily. At Day 63 which is parturition day, plasma progesterone concentration was decreased below 1.0ng/ml with $0.8{\pm}0.2ng/ml$ in pregnant bitches whereas in the non-pregnant bitches at Day 75 were decreased below 1.0ng/ml with 40.5{\pm}0.4ng/ml$. Plasma progesterone concentrations was maintained below 1.0ng/ml during anestrus in all of them. The plasma estradiol-17 $\beta$ concentration was increased above 1.0 pg/ml at the first day of vulval bleeding and it showed a peak Day-1 with 38.2 pg/ml. Thereafter it was sharply decreased after Day 0, which was the day that plasma progesterone concentration was first increase above 4.0ng/ml, and was maintained below basal levels. In relationship between CI and reproductive hormones, plasma estradiol-17 $\beta$concentration showed a peak at Day-3 and CI showed a peak at Day-1 which was the second day after plasma estradiol-17 $\beta$ peak, and plasma progesterone concentration was first increased above 4.0ng/ml at Day 0 which was the first day after CI peak. CI was first increased above 80% at Day-6 which was the third day before plasma estradio-17 $\beta$ peak and it was maintained above 80% between Days-6 and Day 3 during 10 days, and showed above 90% at Day-3 which was the day that plasma estradiol-17 $\beta$peak and was maintained above 90% between Day-3 and Day 3 during 7 days. In conclusion, ovulation in Koran Jin-do bitches occurred at the first day after CI peak, at the third day after plasma estradiol-17 $\beta$peak and the day when plasma progesterone concentration was first increased above 4.0 ng/ml. And it was estimated that the optimal mating time was the day when the CI was maintained above 90% and plasma progesterone concentration was between 3.0~8.0ng/ml. Therefore plasma progesterone concentration measurement was used for determination of an accurate ovulation time and the optimal mating time but also vaginal cytology, which is low-priced with equipment and is the simple examination method, was reliable method for estimating estrous cycle, optimal breeding time and ovulation time in Korea Jin-do bitches.

• Journal of Veterinary Clinics
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• v.31 no.5
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• pp.394-402
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• 2014

The objective of this study was to investigate the effect of chronic unpredictable stress on the reproductive function and ovarian luteinizing hormone receptor (LHR) expression. 9-week-old C57BL/6 female mice were randomly divided into two groups: control group and stressed group. Mice have been stressed twice a day for 35 days with 12 different stressors which were randomly selected. The results demonstrate that there is significant increase in the anxiety-related behaviors (P < 0.05), decrease body weight gain rate (P < 0.01) and decrease in the average of litter size in stressed mice compared with control group (P < 0.01). Furthermore, the rate of primary, secondary and early antral follicles in stressed mice significantly decreased (P < 0.05), whereas that of atretic follicles significantly increased compared with control mice (P < 0.01). The immunohistochemical analysis revealed that reduced LHR expression in granulosa cells of follicle and luteal cells of corpus luteum in response to chronic unpredictable stress. The western blot analysis revealed significantly decrease in LHR expression in the stressed mice ovaries compared with the control (P < 0.05). These results suggest that ovarian LHR expression affected by chronic unpredictable stress and the modulated ovarian LHR is responsible for ovarian follicular maldevelopment and reproductive dysfunction.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.17 no.5
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• pp.423-435
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• 1984

The reproductive cycle of the small filefish, Rudarius ercodes was investigated based on the annual variations of gonadosomatic index(GSI) and hepatosomatic index(HSI) by electronic and photic microscophy. The specimens used were collected at the coastal area of Benden island, Sizuokagen, Japan, from September 1982 to August 1983. GSI began to increase from March, starting season of longer daylength and higher water temperature, and reached the maximum value between June and August. It began to decrease from September with the lowest value appearing between November and February without any evident variation. The annual variations of HSI were not distinct in male filefish and were negatively related to GSI in female : HSI decreased in the summer season when the ovary was getting mature and reached the maximum in the winter season when the ovary was getting retrogressive. The ovary consisted of a pair of saccular structure with numerous ovarian sacs branched toward the median cavity. Oogonia divided and proliferated along the germinal epithelium of the ovarian sac. Young oocytes with basophile cytoplasm showed several scattering nucleoli along the nuclear membrane. when the oocytes growing to about 300 ${\mu}m$, nuclear membrane to disappear with nucleus migrating toward the animal pole. The regions of protoplasm were extremely confined within the animal hemisphere in which most of cytoplasms were filled with yolk materials and oil drops. After ovulation, residual follicles and growing oocytes remaining in the ovarian sacs degenerated. But perinucleatic young oocytes without follicles formed were not degenerated, and growing continuously still in the next year. Mitochondria and endoplasmic reticula in the cytoplasm remarkably increased with oocytes maturing and yolk accumulating. Those were considered to be functionally related to the yolk accumulation. Five or six layers of possible vitellogenin, oval-shaped disc structures with high electron density, appeared in the apex of follicular processes stretching to the microvilli pits of mature oocytes. Testis consisting of a pair of lobular structures in the right and left were united in the posterior seminal vesicle, Cortex of testis was composed of several seminiferous tubules, and medulla consisting of many sperm ducts connected with tubules. Steroid hormone-secreting cells with numerous endoplasmic reticula and large mitochondria of well developed cristae were recognized in the interstitial cells of the growing testis. Axial filament of spermatozoon invaginated deeply in the central cavity of the nucleus and the head formed U-shape with acrosome severely lacking, mitochondria formed large globular paranuclei at the posterior head, and microtubular axoneme of the tail represented 9+9+2 type. The annual reproductive cycles could be divided into five successive stages : growth(March to July), maturation(May to September), Spawning(mid May to early October) and resting stages(October to February). The spawning peak occurred from June to August.