Effect of Exposure Duration of Ovaries and Oocytes at Ambient Temperature on Parthenogenetic Development of Porcine Follicular Oocytes

KIM, Hyun-Jong,CHOI, Sun-Ho,SON, Dong-Soo,CHO, Sang-Rae,CHOE, Chang-Yong,KIM, Young-Keun,HAN, Man-Hye,RYU, Il-Sun,KIM, In-Cheul,KIM, Il-Hwa,IM, Kyung-Soon,NAGAI, Takashi Society for Reproduction and Development 2006 The Journal of reproduction and development Vol.52 No.5

<P>This study was conducted to evaluate the effects of exposing porcine ovaries to 30-33 C during transportation for 4 h and subsequently room temperature (25 C) for 6 h of storage on <I>in vitro</I> maturation (IVM) and subsequent parthenogenetic development of oocytes collected from the ovaries. After IVM, oocytes having a tight oopalsm membrane and no signs of degeneration were exposed to Dulbecco's phosphate-buffered saline (DPBS) with 7% ethanol (v/v) for 7 min to induce parthenogenetic activation. Moreover, we also determined whether exposure of the collected oocytes to room temperature for 1, 2 and 4 h in DPBS or porcine follicular fluid (pFF) affected parthenogenetic development. When porcine ovaries were stored after transportation, oocytes collected from the stored ovaries showed a significantly higher rate of degeneration after 65 h of IVM (58.4%) and a significantly lower rate of cleavage after parthenogenetic activation (40.1%) than oocytes collected from ovaries immediately after transportation (38.9% and 47.4%, respectively). However, there was no significant difference in developmental rates to the morula and blastocyst stages between these two groups (14.4% and 14.3%, respectively). The duration of preservation, 1, 2, and 4 h, of oocytes in DPBS did not affect parthenogenetic development. In contrast, when preserved for 4 h in pFF, the developmental rates of the oocytes were significantly decreased. This suggested that some factor(s) in follicular fluid affects the developmental rate of oocytes with the passage of time in ambient conditions. These results suggest that even after 6 h storage of ovaries, oocytes having normal morphology after IVM have the same rate of parthenogenetic development as oocytes collected from ovaries just after 4 h of transportation, except for a lower cleavage rate, and that exposure of oocytes to room temperature for 4 h in DPBS does not affect their parthenogenetic developmental competence.</P>

Estrogen Receptors are Involved in Xenoestrogen Induction of Growth Hormone in the Rat Pituitary Gland

DANG, Vu Hoang,CHOI, Kyung-Chul,JEUNG, Eui-Bae Society for Reproduction and Development 2009 Journal of Reproduction and Development Vol.55 No.2

<P>Growth hormone (GH) plays a pivotal role in the regulation of growth, development and body composition. In order to provide new insights into estrogenic endocrine disruptor (ED) activities in the pituitary gland and the potential role played by estrogen receptors (ERs) in mediating their effects <I>in vivo</I>, we examined <I>GH</I> expression in the pituitary gland of an immature rat model. At postnatal day 14, immature rats were treated with various doses of <I>4-tert</I>-octylphenol (OP), <I>p</I>-nonylphenol (NP) and bisphenol A (BPA), and the <I>GH</I> mRNA and protein expression levels were analyzed by real-time quantitative PCR and western blot/immunohistochemistry (IHC), respectively. An anti-estrogen (ICI 182780) was used to examine the potential involvement of ERs in ED-induced GH expression during critical windows of development. <I>GH</I> mRNA expression increased significantly 48 h after treatment with a high dose (600 mg/kg body weight [BW]) of OP or NP. However, this induction was abolished completely by co-treatment with ICI 182780. No significant difference in <I>GH</I> mRNA expression was observed following treatment with BPA or co-treatment of BPA with the anti-estrogen. Exposure to high doses (600 mg/kg BW) of these EDs significantly enhanced GH protein expression in the rat pituitary gland, whereas pretreatment with ICI 182780 markedly reduced this expression. Taken together, we have demonstrated for the first time that <I>in vivo</I> exposure to EDs can induce <I>GH</I> mRNA and protein expression in the rat pituitary gland and that their activities may involve an ER-mediated signaling pathway. These results may provide critical evidence for ED-induced dysregulation of pituitary GH expression and thus may be important for elucidating the potential impacts of EDs in altered body growth and development and for predicting the health risks of ED exposure in humans and wildlife.</P>

Junction-mediating and regulatory protein (JMY) is essential for early porcine embryonic development

LIN, Zi Li,CUI, Xiang-Shun,NAMGOONG, Suk,KIM, Nam-Hyung The Society for Reproduction and Development 2015 The Journal of reproduction and development Vol.61 No.5

<P> Junction-mediating and regulatory protein (JMY) is a regulator of both transcription and actin filament assembly. JMY is a critical nucleation-promoting factor (NPF); however, its role in the development of mammalian embryos is poorly understood. In the current study, we investigated the functional roles of the NPF JMY in porcine embryos. Porcine embryos expressed JMY mRNA and protein, and JMY protein moved from the cytoplasm to the nucleus at later embryonic developmental stages. Knockdown of JMY by RNA interference markedly decreased the rate of blastocyst development, validating its role in the development of porcine embryos. Furthermore, injection of JMY dsRNA also impaired actin and Arp2 expression, and co-injection of actin and Arp2 mRNA partially rescued blastocyst development. Taken together, our results show that the NPF JMY is involved in the development of porcine embryos by regulating the NPF-Arp2-actin pathway.</P>

Conservation of Migration and Differentiation Circuits in Primordial Germ Cells Between Avian Species

PARK, Tae Sub,HAN, Jae Yong The Society for Reproduction and Development 2013 The Journal of reproduction and development Vol.59 No.3

<P><B>Abstract </B></P><P> Germ cell differentiation in reverse-sexed reproductive organs and interspecies germ line chimeras provides insight into the mechanism of germ cell development and represents a useful tool for conservation of endangered birds. We investigated the migration and survival capacity of male chicken primordial germ cells (PGCs) in female chicken embryos and in quail and Korean ring-necked pheasant embryos of both sexes. Interestingly, the PGCs were successfully reintroduced in all cases. Furthermore, the cells survived in the recipient gonads until hatching regardless of sex and species of the recipient. In the case of male recipient chickens, PGC-derived offspring were produced. However, the reverse-sexed female chickens, quails and pheasants of both sexes did not generate any male donor PGC-derived progeny. These results suggest that migration and survival circuits in chicken PGCs are conserved in both sexes and between avian species during embryonic development.</P>

Risk Factors and the Economic Impact of Ovarian Cysts on Reproductive Performance of Dairy Cows in Korea

KIM, Ki-Doek,KI, Kwang-Sook,KANG, Hyun-Gu,KIM, Ill-Hwa Society for Reproduction and Development 2005 The Journal of reproduction and development Vol.51 No.4

<P>The objectives of this study were to determine the risk factors for development of postpartum ovarian cysts by evaluating several reproductive factors in individual cows, and to determine the economic impact of ovarian cysts on subsequent reproductive performance in dairy herds in Korea. The data, including cow parity, abnormal puerperium, endometritis, body condition score (BCS), and breeding status were collected from 634 cows in 9 dairy herds. We used logistic regression to evaluate the effects of these factors on ovarian cysts. A stepwise procedure, used to obtain the appropriate model with α=0.05, revealed that cow parity was the most important risk factor for ovarian cyst development within 8 weeks postpartum, while development of endometritis and BCS loss ≥1 from the dry period to 8 weeks postpartum were the most important risk factors for ovarian cyst development beyond 8 weeks postpartum. The occurrence of ovarian cysts beyond 8 weeks postpartum prolonged (P<0.01) the mean intervals from calving to first service (27 days) and conception (77 days), and increased (P<0.05) the culling rate (7.8%), while ovarian cyst development within 8 weeks postpartum did not affect (P>0.05) the mean intervals from calving to first service and conception or the culling rate. The economic loss resulting from the occurrence of ovarian cysts was estimated at approximately 823,996 won ($687) due to effects on the cost of nutrition, average growth of calves, labor and medical costs, and culling. These results suggest that cow parity is correlated with the development of ovarian cysts within 8 weeks postpartum, and endometritis and BCS loss ≥1 from the dry period to 8 weeks postpartum are correlated with the development of ovarian cysts after 8 weeks postpartum, which decreases reproductive performance and results in economic loss in dairy herds in Korea.</P>

Differential Effects of Flutamide and Di-(2-ethylhexyl) phthalate on Male Reproductive Organs in a Rat Model

VO, Thuy T. B.,JUNG, Eui-Man,DANG, Vu Hoang,JUNG, Kikyung,BAEK, Jounghee,CHOI, Kyung-Chul,JEUNG, Eui-Bae Society for Reproduction and Development 2009 Journal of Reproduction and Development Vol.55 No.4

<P>Endocrine disruptors (EDs) with androgenic and anti-androgenic effects may alter reproductive function by binding to androgenic receptors (AR) and inducing or modulating AR-dependent responses in the male reproductive system. However, the molecular mechanism(s) underlying these events remains unclear. In the present study, pregnant Sprague Dawley (SD) rats were treated with testosterone propionate (TP), flutamide (Flu) and di-(2-ethylhexyl) phthalate (DEHP) from gestation days (GD) 11 to 21. Interestingly, maternal exposure to Flu or DEHP caused fluctuations in the neonatal levels of serum testosterone (T) and luteinizing hormone (LH). Serum testosterone and LH were upregulated by Flu, but these hormones were down-regulated by DEHP. The anogenital distances (AGD) of male newborns were determined at post-neonatal days (PND) 1, 21 and 63. Male rats treated prenatally with DEHP (100 mg/kg mother's body weight) or Flu showed an AGD shorter than that of control rats. At PND 63, sperm concentration, viability and motility were reduced in the maternal DEHP and Flu-treated groups. The numbers of seminiferous tubules were reduced in the Flu and DEHP-treated offspring when compared with the vehicle- and TP-treated groups, and the tubules of the testes at PND 63 were disrupted by a high dose of Flu. In addition, we found differential gene expression patterns by microarray analysis following ED exposure, particularly in sex determination-related genes. Although Flu and DEHP are considered to be identical with regard to their anti-androgenic effects, their effects on developing male reproductive organs were distinct, suggesting that Flu competes with endogenous T, while DEHP influences a different step in androgenesis.</P>

Effects of Postnatal Administration of Diethylstilbestrol on Puberty and Thyroid Function in Male Rats

SHIN, Jae-Ho,KIM, Tae Sung,KANG, Il Hyun,KANG, Tae Seok,MOON, Hyun Ju,HAN, Soon-Young Society for Reproduction and Development 2009 Journal of Reproduction and Development Vol.55 No.5

<P>To examine the effects of diethylstilbestrol (DES) on male pubertal development and thyroid function, juvenile male Sprague-Dawley rats were given DES daily by oral intubation at doses of 10, 20 and 40 μg/kg/day from postnatal day 33 for 20 days. Prepuce separation was significantly delayed at the dose of 20 μg/kg/day and above in the DES-treated rats. DES treatment induced a significant reduction in the weights of testes, epididymides, the ventral prostate, seminal vesicles plus coagulating glands and fluid, levator ani bulbocavernosus muscles, Cowper's glands and the glans penis. The weights of the liver and adrenals increased in the DES-treated animals. DES caused a dose-dependent reduction in germ cells; in particular the spermatids were mainly affected. The serum levels of testosterone and luteinizing hormone were significantly reduced in the DES-treated groups, but that of estradiol decreased. No differences were observed in the serum thyroxine levels of the control and DES-treated groups. In microscopic observation of the DES-treated animals, degeneration of germ cells and tubular atrophy in the testis were noted, but there were no microscopic changes in the thyroid. These results indicate that DES affected the pubertal development of juvenile male rats and that its mode of action may be related to alterations in hormone levels.</P>

Effect of the Polyvinylpyrrolidone Concentration of Cryoprotectant on Mouse Embryo Development and Production of Pups: 7.5% of PVP is Beneficial for <i>In Vitro</i> and <i>In Vivo</i> Development of Frozen-Thawed Mouse Embryos

KIM, Chi-Gu,YONG, Hwanyul,LEE, Gene,CHO, Jaejin Society for Reproduction and Development 2008 Journal of Reproduction and Development Vol.54 No.4

<P>In this study, we investigated the effect of polyvinylpyrrolidone (PVP) concentration on <I>in vitro</I> and <I>in vivo</I> development of 2 cell stage, vitrified ICR mouse embryos using a cryoprotectant consisting of ethylene glycol (EG) and sucrose. M2 was selected as the basic medium for vitrification and thawing. After equilibration with 4% (v/v) EG at 37 C for 15 min, the embryos were vitrified with 35% EG, 5, 6 or 7.5% (w/v) PVP and 0.4 M sucrose at 37 C for 30 sec. One week later, the cryotubes of cryopreserved embryos in liquid nitrogen were directly immersed into a 37 C water bath for 1 min and transferred serially into 300 <I>μ</I>l of 0.5 or 0.3 M sucrose at room temperature for 5 min and M2 medium at 37 C for 10 min. The surviving embryos were cultured in KSOM (potassium simplex optimized medium) for 96-120 h in an atmosphere of 5% CO<SUB>2</SUB> in humidified air. Survival was evaluated by morphological appearance, including membrane integrity and presence of apoptotic blastomeres after thawing. For <I>in vivo</I> evaluation, blastocysts were transferred to the uteri of pseudopregnant mice. The survival rates of the 5 and 7.5% PVP concentration groups showed a significantly higher difference compared with that of the 6% PVP group (85.5 and 86.5 <I>vs.</I> 71.2%), respectively. Each pup in the of 5 and 6% groups was cannibalized immediately after parturition. A litter of live pups was obtained from only the 7.5% PVP groups. Our study indicated that supplementation of EG and sucrose cryoprotectant solution with 7.5% PVP is optimal for successful vitrification of 2-cell stage ICR mouse embryos.</P>

Differential Apoptotic and Proliferative Activities of Wild-type FOXL2 and Blepharophimosis-ptosis-epicanthus Inversus Syndrome (BPES)-associated Mutant FOXL2 Proteins

KIM, Jae-Hong,BAE, Jeehyeon The Society for Reproduction and Development 2014 The Journal of reproduction and development Vol.60 No.1

<P><B>Abstract</B></P><P> FOXL2 is an essential transcription factor that is required for proper development of the ovary and eyelid. Mutations in FOXL2 cause an autosomal dominant genetic disorder, blepharophimosis-ptosis-epicanthus inversus syndrome (BPES). BPES type I patients have eyelid malformation and premature ovarian failure leading to infertility, whereas women with type II BPES are fertile or subfertile. In the present study, we evaluated and compared apoptotic and antiproliferative activities of wild-type (WT) and mutant FOXL2 proteins found in BPES type I and II in human granulosa cell tumor-derived KGN cells. Ectopic expression of WT FOXL2 induced apoptosis and inhibited cell cycle progression in human granulosa cells. In contrast, mutated FOXL2s found in BPES type I significantly reduced these activities, whereas mutated FOXL2s in BPES type II showed intermediate activities. Furthermore, mutant FOX L2 proteins were defective in activating transcription of target genes including <I>Caspase 8</I>, <I>TNF-R1</I>, <I>FAS</I>, <I>p21</I>, and <I>BMP4</I>, which regulate apoptosis, proliferation, and differentiation of granulosa cells. Thus, decreased apoptotic and antiproliferative activities caused by mutant forms of FOXL2 found in BPES patients may at least partially contribute to the pathophysiology of ovarian dysfunction.</P>

Absence of nucleolus formation in raccoon dog-porcine interspecies somatic cell nuclear transfer embryos results in embryonic developmental failure

JEON, Yubyeol,NAM, Yeong-Hee,CHEONG, Seung-A,KWAK, Seong-Sung,LEE, Eunsong,HYUN, Sang-Hwan The Society for Reproduction and Development 2016 The Journal of reproduction and development Vol.62 No.4

<P> Interspecies somatic cell nuclear transfer (iSCNT) can be a solution for preservation of endangered species that have limited oocytes. It has been reported that blastocyst production by iSCNT is successful even if the genetic distances between donors and recipients are large. In particular, domestic pig oocytes can support the development of canine to porcine iSCNT embryos. Therefore, we examined whether porcine oocytes may be suitable recipient oocytes for Korean raccoon dog iSCNT. We investigated the effects of trichostatin A (TSA) treatment on iSCNT embryo developmental patterns and nucleolus formation. Enucleated porcine oocytes were fused with raccoon dog fibroblasts by electrofusion and cleavage, and blastocyst development and nucleolus formation were evaluated. To our knowledge, this study is the first in which raccoon dog iSCNT was performed using porcine oocytes; we found that 68.5% of 158 iSCNT embryos had the ability to cleave. However, these iSCNT embryos did not develop past the 4-cell stage. Treatment with TSA did not affect iSCNT embryonic development; moreover, the nuclei failed to form nucleoli at 48 and 72 h post-activation (hpa). In contrast, pig SCNT embryos of the control group showed 18.8% and 87.9% nucleolus formation at 48 and 72 hpa, respectively. Our results demonstrated that porcine cytoplasts efficiently supported the development of raccoon dog iSCNT embryos to the 4-cell stage, the stage of porcine embryonic genome activation (EGA); however, these embryos failed to reach the blastocyst stage and showed defects in nucleolus formation.</P>