• Animal Bioscience
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• v.35 no.1
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• pp.75-86
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• 2022

Objective: The objective of this experiment was to investigate the effect of dietary glucose oxidase (GOD), catalase (CAT), or both supplementation on reproductive performance, oxidative stress, and apoptosis in sows. Methods: A total of 104 multiparous sows were randomly assigned to four groups (n = 26) with each group given a basal diet, basal diet plus GOD at 60 U/kg, basal diet plus CAT at 75 U/kg, and basal diet plus GOD at 60 U/kg and CAT at 75 U/kg. Sows were fed the experimental diets throughout gestation and lactation. Results: Dietary GOD supplementation increased average daily feed intake of sows and litter weight at weaning (p<0.05). Dietary CAT supplementation reduced the duration of parturition, stillbirth, and piglet mortality and increased growth performance of weaned piglets (p<0.05). Dietary GOD and CAT supplementation enhanced antioxidant enzyme activities and lessened oxidative stress product levels in plasma of sows and elevated antioxidant capacity of 14-day milk and plasma in weaned piglets (p<0.05). Dietary GOD supplementation increased fecal Lactobacillus counts and reduced Escherichia coli counts of sows (p<0.05). Compared with the basal diet, the GOD diet reduced fecal Escherichia coli counts of sows, but the addition of CAT did not reduce Escherichia coli counts in the GOD diet. Dietary GOD and CAT supplementation reduced the apoptosis rate of the liver, endometrium, and ovarian granulosa cells in sows (p<0.05). In the liver, uterus, and ovary of sows, the mRNA expression of caspase-3 and caspase-9 was downregulated by dietary GOD and CAT supplementation (p<0.05). Conclusion: Dietary GOD and CAT supplementation could improve the antioxidant capacity of sows and weaned piglets, and alleviate hepatic, ovarian and uterine apoptosis by weakening apoptosis-related gene expression. Glucose oxidase regulated fecal microflora of sows, but supplementation of CAT to GOD could weaken the inhibitory effect of GOD on fecal Escherichia coli.

• Development and Reproduction
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• v.8 no.1
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• pp.57-64
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• 2004

This study was carried out to examine the expression of the circadian clock genes in the mouse ovary and testis at different developmental stages. Expression of Period1(Per 1), Period2(Per2), Period3(Per3), Cryptochrome1(Cry1), Cyptochrome2(Cry2), Clock Small and Prokineticin1 and Prokineticin2 receptor(Prok1r, Prok2r) genes in mouse ovary was explored by semiquantitative reverse transcription Polymerase chain reaction(RT-PCR) according to the developmental stage(post partum day; ppd 1, 7, 10, 21 and 35). Immunohistochemistry using PER1 antibody was also analyzed. The differential expression pattern of clock genes was presented according to stages of the mouse ovarian development (ppd 1, 7, 10, 21 and 35). In the cases of ovaries, at the starting point of follicle growth at ppd 7 and 10, the clock gene expression patterns were changed vastly. According to the developmental stages, the clock genes were highly expressed at ppd 7 and 10 in mouse testis also. Receptors for Prok2, the circadian output molecule of SCN, were also expressed in ovary at ppd 7 and in testis at ppd 1 and 7, respectively. Immnunohistochemical analysis of PER1 showed positive signals in the cytoplasm of oocytes and granulosa cells. The level or PER1 expression was increased in cells at the spermatogonia and the condensing spermatids. The expression pattern of Perl and localization of PER1 were showed similar patterns according to the developmental stages in ovary and testis. Taken together, it could be observed that the expression of clock genes was highly correlated with gonadal development and germ cell differentiation in mice. Therefore, in this study, circadian programming of the genes in the ovary and testis is strongly imposed across a wide range of core reproductive cycles and normal development of gametes. Although the existence of circadian genes is clearly investigated, further studies on the direct evidence is required for the understanding of the relationship between circadian genes and regulation of gonadal differentiation and germ cell development.

• 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.

• Applied Microscopy
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• v.31 no.1
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• pp.19-35
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• 2001

Outlines for plasma $estradiol-17\beta$, components, electrophoretic patterns, and ultrastructural changes were obtained in female rainbow trout (Oncorhynchus mykiss) during the seasonal reproductive cycles. Plasma $estradiol-17\beta$ under the natural conditions, exhibited distinct seasonal variation, peaking very late in vitellogenic season during September, decreasing gradually the halt of spawning in December, and ultimately falling during the early stages of seasonal ovarian recrudescence in February and March. This change in $estradiol-17\beta$ appeared to stimulate vitellogenin production as evidenced by increases in plasma calcium, phosphorus, glucose, albumin and total protein levels. The electrophoretic patterns of late maturing or spawning oocytes were stained more intensively than those of late perinucleolus oocytes (molecular weights of approximately 70,000 and 200,000). Two protein bands were found in the SDS-PAGE separation, coincident with the $estradiol-17\beta$ hormone peak. Gonadosomatic indices (GSI) significantly increased from October to January, and showed the highest peak in January, coinciding with the numerically abrupt increase of ripe ova in female. A positive correlation (r=0.701, p<0.01) was established between plasma $estradiol-17\beta$ levels and the gonadosomatic index during the prespawning. The highest level of hepatosomatic index (HSI) observed in December. During the breeding season (December), the gonadotropes were large and filled with GTH-containing inclusions such as granules and globules. The vitellogenic phase began as late perinurleolus oocytes became transformed into early maturing oocytes through the accumulation of yolk, and oocytes reached the late maturing stages as the ooplasm was completely packed with yolk. Marked ultrastructural changed in the granulosa cells during nuclear migration involve the dilation of the rough endoplasmic reticulum and the appearance of the rod-shaped mitochondria with tubular cristae. Microvilli (finger-like projections), from the zona radiata and from the oocyte grew, and made contact with each other in the pore canals of the zona radials during vitellogenesis, but were withdrawn as the zona radiata became more compact and devoid of pore canals during oocyte maturation. The zona radiata grew to a tripartite structure such as an outer thin homogeneous layer, and two inner thick helicoidal layers (zona radials interna and zona radiata externa). Under the normal conditions, the ovarian follicle influenced the histological development and periodical secretion of the hormones , sufficient for a oogenesis and gonadal steroid production.

• Clinical and Experimental Reproductive Medicine
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• v.34 no.2
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• pp.95-106
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• 2007

Objective: We previously identified differentially expressed genes (DEGs) between germinal vesicle (GV) and metaphase II (MII) mouse oocyte. The present study was accomplished as a preliminary study to elucidate the role of ribose 5-phosphate isomerase A (Rpia), the essential enzyme of the pentose phosphate pathway (PPP), in oocyte maturation. We observed expression of Rpia in the mouse and porcine oocytes. Methods: Expression pattern of the 11 MII-selective DEGs in various tissues was evaluated using RT-PCR and selected 4 genes highly expressed in the ovary. According to the oocyte-selective expression profile, we selected Rpia as a target for this study. We identified the porcine Rpia sequence using EST clustering technique, since it is not yet registered in public databases. Results: The extended porcine Rpia nucleotide sequence was submitted and registered to GenBank (accession number EF213106). We prepared primers for porcine Rpia according to this sequence. In contrast to the oocyte-specific expression in the mouse, Rpia was expressed in porcine cumulus and granulosa cells as well as in oocytes. Conclusion: This is the first report on the characterization of the Rpia gene in the mouse and porcine ovarian cells. Results of the present study suggest that the mouse and porcine COCs employ different mechanism of glucose metabolism. Therefore, the different metabolic pathways during in vitro oocyte maturation (IVM) in different species may lead different maturation rates. It is required to study further regarding the role of Rpia in glucose metabolism of oocytes and follicular cell fore exploring the regulatory mechanism of oocyte maturation as well as for finding the finest culture conditions for in vitro maturation.

• Development and Reproduction
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• v.10 no.2
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• pp.147-154
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• 2006

Phthalates such as di(2-ethyl hexyl)phthalate(DEHP) are industrial chemicals with wide-ranging human exposures because of their use in plastics and other common consumer products. Consequently, their adverse effects as endocrine disruptor in the reproductive physiology of both laboratory rodents and human have been studied extensively. The present study was undertaken to examine whether prepubertal exposure to DEHP affects on the onset of puberty and the associated reproductive parameters such as hormone receptor expressions in female rats. DEHP(100mg/kg/day) was administered daily from postnatal day 25(PND 25) through the day when the first vaginal opening(VO) was observed, and the animals were sacrificed on the next day. Gross anatomy and weight of reproductive tissues were compared to test the DEHP's effects on the cell proliferation. Furthermore, histological studies were performed to assess the structural alterations in the tissues. Specific radioimmunoassay was carried out to measure serum LH levels. To determine the transcriptional changes in progesterone receptor(PR), total RNAs were extracted and applied to the semi-quantitative reverse transcription polymerase chain reaction(RT-PCR). As a result, delayed VO was shown in the DEHP group(PND $37.3{\pm}0.7$) compared to the control group(PND $35.3{\pm}0.7$; p<0.05). DEHP treatment significantly decreased the wet weight of ovaries and uteri compared to the control group(p<0.05). Interestingly, elevation of serum LH levels was shown in the DEHP group(p<0.05). Graafian follicles and corpora lutea were observed only in the ovaries from the control animals. Numerous primary, secondary follicles and small atretic follicles were observed in the ovaries from DEHP-treated animals. Similarly, hypotrophy of luminal and glandular uterine epithelium was found in the DEHP-treated group. These effects were probably due to the inhibitory effects of DEHP on the synthesis and secretion of estrogen from granulosa cells. In the semiquantitative RT-PCR studies, the transcriptional activities of PR in both ovary(p<0.05) and uterus(p<0.01) from DEHP-treated animals were significantly lower than those from the control animals. The present studies demonstrated that the acute exposure to DEHP during the critical period of prepubertal stage could inactivate the reproductive system resulting delayed puberty in female rats.

• Journal of Embryo Transfer
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• v.21 no.2
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• pp.157-162
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• 2006

It is well known that unidentified factors in sera, hormones and growth factors promote the proliferation of granulosa cells and nuclear maturation of bovine COCs (cumulus oocytes complexes) in vitro. Attempts had been developed the simple composition of culture media and similar system to in vivo conditions has been applied. In the present study, we investigated the effect of FGF (fibroblast growth factor) on in vitro maturation and in vitro development of Hanwoo COCs. When the COCs were matured in HPM 199 (Inst. of Functional peptide, Japan) containing 0.1, 1 and 10 ng/ml FGF for 24 hr, maturation rates to metaphase II ($70.0{\sim}75.0%$) were significantly higher (p<0.05) than that of control group (0 ng/ml FGF, 37.5%). When matured COCs with FGF were cultured in maturation medium after in vitro fertilization, developmental rates to blastocysts were 9.5, 0 and 2.9%, respectively, compared to 25.0% of the control group (p<0.05). When the matured COCs with FGF were cultured in HPM 199 (IFP971, Inst. of Functional peptide, Japan) containing 10% FBS, 0.8% BSA or 0.1% PVA (polyvinyl alcohol), the blastocyst formation rates were 12.4, 12.8 and 8.5%, respectively, while the rates of matured COCs with FGF and cultured with IVMD and IVD (Inst. of Functional peptide, Japan) without serum were 38.4% and 34.8%, respectively (p<0.05). These results suggested that FGF is available for in vitro maturation of bovine COCs and is not suitable for in vitro development, but further investigation would be need for finding the synergistic autocrine/paracrine fashion of other growth factors in early bovine embryo development.

• Korean Journal of Veterinary Research
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• v.37 no.1
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• pp.71-78
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• 1997

This study was designed to investigate the number of the growing and mature follicles following gonadotrophin treatments for superovulation in mature rats. Eighteen mature rats (Sprague-Duwely, initially 190~230gm) were randomly alloted into 3 groups. One group was control group, another FSH-treated group was injected intramuscularly with 0.5 units of follicular stimulating hormone (FSH) / rat, and third PMS and HCG-treated group was intramuscularly injected with 20~25IU of pregnant mare serum (PMS) / rat and then at the 48 hrs later, with 20~25IU of human chorionic gonadotrophin (HCG) / rat. The uteri and ovaries of rats were collected and then were observed grossly and serial sections of paraffin embedding ovaries were stained with H-E. Number of ovarian follicles by following 3 grades of large, middle and small follicles from secondary and tertiary follicles were investigated by LM photography of preparations. Small follicles were classified as secondary follicles of preantral follicles with more than 2 layers of granulosa cells surrounding the oocyte and middle follicles were classified as secondary follicles with early signs of antral cavity or with more than one small cavity on either side of the oocytes and large follicles were classified as tertiary follicles with a single medium sized antral cavity or large well-formed antral cavity. In gross findings, the uteri were slightly swelling in FSH-treated group and markedly swelling or filled with fluid in the uterine lumen in PMS and HCG-treated group. In histological findings, the shape and size of the follicles were diverse in middle and large follicles of FSH-treated group and PMS and HCG-treated group, and proportion of atretic follicles was increased in FSH-treated group and PMS and HCG-treated group than those in control group. The uteri of FSH-treated group and PMS and HCG-treated group were hypertropied or filled with fluid in the lumens and walls of uteri. The wall tissue layers were flattened and their blood and lymph vessels were dilated. The mean number of follicle per ovary in control group were appeared to be $17.1{\pm}5.6$($14.0%{\pm}4.6%$), $37.8{\pm}9.1$($30.9{\pm}7.4%$) and $67.6{\pm}30.1$($55.2{\pm}24.6%$) respectively at large, middle and small follicles and total number of these 3 grade follicles were appeared to be $122.5{\pm}40.0$. The mean number of follicle per ovary in FSH-treated group were appeared to be $22.8{\pm}7.0$($17.4%{\pm}5.3%$), $43.4{\pm}6.6$($33.2{\pm}5.1%$) and $64.5{\pm}13.0$($49.3{\pm}9.9%$) respectively at large, middle and small follicles and total number of these 3 grade follicles were appeared to be $130.7{\pm}16.6$. The mean number of follicle per ovary in PMS and HCG-treated group were appeared to be $29.7{\pm}11.0$($16.3%{\pm}6.0%$), $61.9{\pm}17.2$($33.9{\pm}9.4%$) and $91.1{\pm}28.2$($49.9{\pm}15.4%$) respectively at large, middle and small follicles and total number of these 3 grade follicles were appeared to be $182.6{\pm}32.7$. The above findings reveal that large follicles were increased 29.8% in FSH-treated group and 73.7% in PMS and HCG-treated group than those in control group and in histologic findings, proportion of atretic follicles were more increased in ovaries with more number of more developing follicles.

• Development and Reproduction
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• v.8 no.2
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• pp.119-122
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• 2004

It is well known that unidentified factors in sera, hormones and growth factors promote the proliferation of granulosa cells and nuclear maturation of bovine COCs in vitro. Attemps had been developed the simple composition of culture media and similar system to in vivo conditions has been applied. In the present study, we investigated the effect of TGF-${\beta}$ on in vitro maturation and in vitro development of Hanwoo COCs. When the COCs were matured in TCM 199 containing 0.1, 1 or 10 ng/ml TGF-${\beta}$ for 24 hrs, metaphaseⅡ of COCs were obtained 95.8%, 100% of matured COCs, respectively and there were no differences among the concentrations of TGF-${\beta}$. Matured COCs with TGF-${\beta}$ cultured in maturation medium after in vitro fertilization, developmental rate to blastocyst were 0~0.8%. Matured COCs with TGF-${\beta}$ were cultured in TCM 199+10% FBS, 0.8% BSA, 0.1% PVA, blastocyst formation were showed in 12.4%, 12.8%, 8.5% of those and cultured in IVMD or IVD without serum were 38.4%, 34.8%, respectively. There were significant differences among the media (P<0.05). TGF-${\beta}$ is available for i vitro maturation of bovine COCs, but further investigation would be need for finding the synergistic autocrine/paracrine fashion of other growth factors in early bovine development.