• Development and Reproduction
• /
• v.17 no.3
• /
• pp.269-274
• /
• 2013

This study was performed to evaluate the effects of fibroblast co-culture on in vitro maturation (IVM) of prepubertal mouse preantral follicles. The intact preantral follicles were obtained from the ovaries of 12-14 day old mice and these were cultured individually in ${\alpha}$-minimal essential medium (${\alpha}$-MEM) supplemented with 5% fetal bovine serum (FBS), $100mIU/m{\ell}$ recombinant follicle stimulating hormone (rFSH), 1% insulin-transferrin-selenium, $100{\mu}g/ml$ penicillin and $50{\mu}g/m{\ell}$ streptomycin as base medium for 12 days. A total of 200 follicles were cultured in base medium co-cultured with mouse embryonic fibroblast (MEF) (MEF group) (n=100) or only base medium as control group (n=100). Survival rate of follicles on day 12 of culture were significantly higher in the MEF group of 90.0%, compared with 77.0% of the control group (p=0.021). Follicle diameters on day 6 and 8 of the culture period were significantly larger in the MEF group than those in the control group (p=0.021, p=0.007, respectively). Estradiol levels in culture media on day 4, 6, 8, 10 and 12 of the culture period were significantly higher in the MEF group (p=0.043, p=0.021, p=0.006, p<0.001 and p=0.008, retrospectively). Our data suggest that MEF cell co-culture on IVM of mouse preantral follicle increases survival rate and promotes follicular growth and steroid production.

• Asian-Australasian Journal of Animal Sciences
• /
• v.20 no.7
• /
• pp.1023-1029
• /
• 2007

Inhibins participate in the regulation of pituitary follicle-stimulating hormone synthesis and secretion, follicular maturation and steroidogenesis in the female. Inhibin ${\beta}_A$ gene (INHBA) was studied as a candidate gene for the prolificacy of sheep. Single nucleotide polymorphisms of the entire coding region and partial 3' untranslated region of INHBA were detected by PCR-SSCP in two high fecundity breeds (Small Tail Han and Hu sheep) and six low fecundity breeds (Dorset, Texel, German Mutton Merino, South African Mutton Merino, Chinese Merino and Corriedale sheep). Only the PCR products amplified by primers 3, 4 and 5 displayed polymorphisms. For primer 3, genotype CC was only detected in Chinese Merino sheep, genotype AA was detected in the other seven sheep breeds. Genotype BB was only detected in Hu sheep. Only Hu sheep displayed polymorphism. Eight or four nucleotide mutations were revealed between BB or CC and AA, respectively, and these mutations did not result in any amino acid change. For primer 4, genotypes EE, EG and GG were detected in Dorset and German Mutton Merino sheep, genotypes EE, EF and FF were detected in Chinese Merino sheep, only genotype EE was detected in the other five sheep breeds. Only Dorset, German Mutton Merino and Chinese Merino sheep displayed polymorphism. Sequencing revealed one nucleotide mutation ($114G{\rightarrow}A$) of exon 2 of INHBA gene between genotype FF and genotype EE, and this mutation did not cause any amino acid change. Another nucleotide change ($143C{\rightarrow}T$) was identified between genotype GG and genotype EE, and this mutation resulted in an amino acid change of $serine{\rightarrow}leucine$. For primer 5, genotypes KK and KL were detected in German Mutton Merino and Corriedale sheep, genotypes KK, LL and KL were detected in the other six sheep breeds. Genotype MM was only detected in Hu sheep. All of these eight sheep breeds displayed polymorphism. Sequencing revealed one nucleotide mutation ($218A{\rightarrow}G$) of exon 2 of the INHBA gene between genotype LL and genotype KK, and nine nucleotide mutations between genotype MM and genotype KK. These mutations did not alter amino acid sequence. The partial sequence (395 bp for exon 1 and 933 bp for exon 2) of the INHBA gene in Small Tail Han sheep (with genotype KK for primer 5) was submitted into GenBank (accession number EF192431). Small Tail Han sheep displayed polymorphisms only in the fragment amplified by primer 5. The Small Tail Han ewes with genotype LL had 0.53 (p<0.05) or 0.63 (p<0.05) more lambs than those with genotype KL or KK, respectively. The Small Tail Han ewes with genotype KL had 0.10 (p>0.05) more lambs than those with genotype KK.

• Proceedings of the Korean Society of Embryo Transfer Conference
• /
• 2002.11a
• /
• pp.113-113
• /
• 2002

The stimulatory effect of EGF and FSH on oocyte maturation have been reported in various mammalian species. And some reports presented FSH enhanced the effect of EGF on oocyte maturation. But, the interaction between EGF and FSH on nuclear maturation of mammalian oocytes is not fully understood. We observed the effect of EGF and FSH on nuclear maturation during in vitro maturation of mouse oocytes. Also, we examined the interaction between EGF and FSH on nuclear maturation of mouse oocytes using the EGFR inhibitor or FSH inhibitor. Germinal vesicle (GV) stage oocytes were obtained from 3-4weeks PMSG primed BCFI hybrid mice and cultured in TCM-199 medium with 0.4%PVP supplemented with/without EGF (1ng/ml), FSH (1ug/ml), EGFR specific tyrosine kinase inhibitors: Tyrphostin AG 1478 (500nM), MAP kinase kinase inhibitor : U0126 (20uM) or PD 98059 (100uM) for 14-l5hr. Rapid staining method were used for the assessment of nuclear maturation. Nuclear maturation rates of EGF indjor FSH-treated group were significantly higher than those of control group. Treatment of EGFR inhibitor significantly block the nuclear maturation of GV oocyte in EGF-treated group, but it did not block those of GV oocyte in FSH-treated or FSH and EGF-treated group. Treatment of FSH inhibitor(U0126, PD98059) significantly block the nuclear maturation of EGF-treated group, FSH-treated and FSH and EGF-treated group. These results show that EGF has a stimulatory effect as well as different action pathway with FSH on in-vitro maturation of mouse oocyte in vitro. Therefore, further studies will be needed to find the signaling pathway of EGF associated with nuclear maturation.

• Korean Journal of Veterinary Research
• /
• v.34 no.2
• /
• pp.395-406
• /
• 1994

To study the conditions to enhance success of embryo transfer in the dog, 20 mixed-breed bitches were used for the experiment along with 4 male dogs for mating. The bitches were paired according to synchronism of natural estrus, or the counterpart as donor or recipient was treated with gonadotropin as FSH (follicular stimulating hormone) or PMSG (pregnant mare serum gonadotropin) for induction of estrus to be synchronized with estrus of the other bitch in natural estrus. Embryo recovery was performed in two ways for comparison, either by flushing each uterine horn after ovariohysterectomy or by flushing each horn in the state of non-ovariohysterectomy. In addition, the result of pregnancy according to the embryo stage and the repeatability of the experimental animals as donor or recipient were also investigated. FSH or PMSG was administered to the bitches which had passed over 4 months from last estrus, resulting in estrus-positive in 3 dogs of 6 FSH-treated dogs (50.0%), and in 5 dogs of 9 PMSG-treated dogs (55.6%), determined by proestrus signs and vaginal smear test. Estrus-positive bitches induced with gonadotropin were used as donor or recipient resulting in one embryo-recovered bitch as donor and one offspring-delivered bitch as recipient in 5 PMSG-treated dogs, whereas no result was obtained from 3 FSH-treated dogs. The rate of embryo recovery to be compared with number of corpus luteum was 68.2% in ovariohysterectomized dogs and 55.2% in non-ovariohysterectomized dogs, respectively. The number of dogs from which embryo was collected were 4 dogs of 6 ovariohysterectomized dogs (66.7%) and 6 dogs of 7 non-ovariohysterectomized dogs (85.7%), respectively. The result of parturition was obtained from one dog of 5 estrus-induced recipients, whereas no result was obtained from 3 natural-estrus recipients. The only dog which delivered a male puppy had been transferred 3 morulae and 2 blastocysts. Of 6 repeat-used bitches in canine embryo transfer, 3 dogs showed repeatability either as donor or recipient. These results indicated that inducing estrus of a dog with gonadotropin is feasible in canine embryo transfer to be synchronized with that of a natural-estrus dog, that embryo recovery is also possible in non-hysterectomized dogs, that the estrus-induced dog is also usable as recipient to result in parturition, and that repeat-use of a bitch as donor or(and) recipient is possible in canine embryo transfer.

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

• Clinical and Experimental Reproductive Medicine
• /
• v.34 no.2
• /
• pp.125-131
• /
• 2007

Objective: The aim of this study was to compare GnRH antagonist and agonist flare-up treatment in the management of poor responder patients. Methods: One hundred forty-four patients from Jan. 1, 2002 to Aug. 31, 2005 undergoing IVF/ICSI treatment who responded poorly to the previous cycle (No. of oocyte retrieved$\leq$5) and had high early follicular phase follicle stimulating hormone (FSH>12 mIU/ml were selected. Seventy-five patients received agonist flare-up protocol and 71 patients received antagonist protocol. We analyzed the number of oocytes retrieved, number of good embryos (GI, GI-1), total dose of hMG administered, implantation rate, cycle cancellation rate, pregnancy rate, live birth rate. Results: The cancellation rate was high in antagonist protocol (53.5% vs. 30.1%). The number of oocyte retrieved, the number of good embyos were high in agonist flare-up group. There was no statistical difference between GnRH agonist flare up protocol and GnRH antagonist protocol in implantation rate (14.5%, 10.1%), clinical pregnancy rate per transfer (29.4%, 21.2%) and live birth rate per transfer (21.6%, 18.2%). Although the result was not statistically significant, GnRH agonist flare up group showed a nearly doubled pregnancy rate and live birth rate per initial cycle than GnRH antagonist group. Conclusions: The agonist flare-up protocol appears to be slightly more effective than the GnRH antagonist protocol in implantation rate, pregnancy rate, live birth rate but shows statistically no significance. Agonist flare-up protocol improved the ovarian response in poor responders. However, based of the result of the study, we can expect improved ovarian response in poor responders by GnRH agonist flare up protocol.