• Clinical and Experimental Reproductive Medicine
• /
• v.28 no.2
• /
• pp.79-86
• /
• 2001

Objective: Mouse pre-antral follicles require the addition of gonadotropins (Gns) to complete maturation and ovulation of oocyte and antrum formation in vitro. However, we tried examination of in vitro growth of mouse pre-antral follicles in medium without Gns and/or phygiological factors. And also, pre-antral follicles were isolated from ovaries by mechanical method. Our present studies were conducted to evaluate on the growth of follicles and intra-follicular oocytes and antrum formation in vitro of mouse pre-antral follicles in two different media. Methods: Pre-antral follicles ($91{\sim}120{\mu}m$) were isolated mechanically by fine 30G needles not using enzymes from ovaries of 3-6 week-old female ICR mice. Isolated pre-antral follicles were cultured in $20{\mu}l$ droplets of TCM (n=17; follicles: $107.8{\pm}1.58{\mu}m$; oocytes: $57.9{\pm}1.2{\mu}m$) or MEM (n=12; follicles: $109.3{\pm}2.53{\mu}m$; oocytes: $55.4{\pm}1.6{\mu}m$) under mineral oil on the 60 mm culture dish. All experimental media was supplemented with 10% FBS without Gns and/or physiological factors. Pre antral follicles were individually cultured for 8 days. Antram formation and growth of pre-antral follicles and intra-follicular oocytes were evaluated using precalibrated ocular micrometer at X200 magnifications during in vitro culture. Results were analyzed using combination of Student's t-test and Chi-square, and considered statistically significant when p<0.05. Results: Antrum formation had started in two culture media on day 2. On day 8, antrum formation had occurred in 58.3% of pre-antral follicles cultured in DMEM, but only in 23.5% of those cultured in TCM (p=0.0364). Growth of pre-antral follicles and intra-follicular oocytes were observed on day 4 and 8. On day 4, follicular diameter was similar (p=0.1338) in TCM ($119.4{\pm}2.58{\mu}m$) and MEM ($125.4{\pm}4.52{\mu}m$). However, on day 8, diameters of pre-antral follicles cultured in MEM ($168.9{\pm}17.29{\mu}m$) were significantly bigger (p=0.0248) than that in TCM ($126.7{\pm}4.28{\mu}m$). On day 4 and 8, diameters of intra-follicular oocytes were similar in TCM ($67.1{\pm}1.3$ and $72.4{\pm}0.9{\mu}m$) and MEM ($65.2{\pm}1.7$ and $73.3{\pm}1.5{\mu}m$), respectively. Conclusion: We can conform that medium without Gns and/or physiological factors can be used for in vitro antrum formation and growth of pre-antral follicles and intra-follicular oocytes in mouse. In conclusion, MEM supplemented with FBS can be used for growth in vitro of mouse pre-antral follicles isolated mechanically.

• Proceedings of the KSAR Conference
• /
• 2001.03a
• /
• pp.8-8
• /
• 2001

Mouse follicles require the addition of gonadotropins (Gns) to complete maturation and ovulation of oocyte and antrum formation of follicles in vitro. However, we tried examination of in vitro growth of mouse pre-antral follicles in medium without Gns and physiological factors. And also, pre-antral follicles were isolated from ovaries by mechanical method. Our present studies were conducted to evaluate on the growth of follicles and intra-follicular oocytes and antrum formation in vitro of mouse pre-antral follicles in two different media. Pre-antral follicles (91-120${\mu}{\textrm}{m}$) were isolated mechanically by fine 30G needles not using enzymes from ovary of 3-6 weeks old female ICR mice. Isolated pre-antral follicles were cultured in 20 ${mu}ell$ droplets of TCM (n=17; follicles: 107.8 $\pm$ 1.58 ${\mu}{\textrm}{m}$; oocytes: 59.9$\pm$1.2 ${\mu}{\textrm}{m}$) or MEM (n=12; follicles: 109.3$\pm$2.53 ${\mu}{\textrm}{m}$; oocytes: 55.4 $\pm$1.6${\mu}{\textrm}{m}$) under mineral oil on the 60mm culture dish. All experimental media was supplemented with 10% FBS but without Gns and/or physiological factors. Pre-antral follicles were individually cultured in drops for 8 days. Antrum formation and growth of pre-antral follicles and intra-follicular oocytes were evaluated using a precalibrated ocular micrometer at $\times$200 magnifications during in vitro culture. Results between different groups were analyzed using combination of Student's t-test and Chi-square, and considered statistically significant when P<0.05. Antrum formation of pre-antral follicles had started in two culture media on day-2. On day-8, antrum formation had occurred in 58.3%(7/12) of pre-antral follicles cultured in MEM, but only in 23.5% (4/17) of those cultured in TCM (P=0.0364). Growth of pre-antral follicles and intra-follicular oocytes were observed on day-4 and -8. On day-4, follicular diameters was similar (P=0.1338) in TCM (119.4$\pm$2.58 ${\mu}{\textrm}{m}$) and MEM (125.4$\pm$4.52 ${\mu}{\textrm}{m}$). However, on day-8, diameters of pre-antral follicles cultured in MEM (168.9$\pm$17.29 ${\mu}{\textrm}{m}$) was significantly (P=0.0248) bigger than that in TCM (126.7$\pm$4.28 ${\mu}{\textrm}{m}$). On day-4 and -8, diameters of intra-follicular oocytes were similar TCM (67.1$\pm$1.3 and 72.4$\pm$0.9${\mu}{\textrm}{m}$) and MEM (65.2$\pm$1.7 and 73.3$\pm$1.5 ${\mu}{\textrm}{m}$), respectively. We can conform that medium not supplemented with Gns and/or physiological factors can be used for in vitro antrum formation and growth of mouse pre-antral follicles and intra-follicular oocytes. In conclusion, MEM supplemented with FBS can be used for growth in vitro of mouse pre-antral follicles isolated mechanically.

• Animal Bioscience
• /
• v.37 no.6
• /
• pp.1007-1020
• /
• 2024

Objective: We increased the nuclear maturation rate of antral follicle derived oocytes by using a pre-in vitro maturation (IVM) culture system and improved the developmental potential of these porcine pathenotes by supplementing with melatonin. Furthermore, we investigated the expression patterns of genes involved in cumulus expansion (HAS2, PTGS2, TNFAIP6, and PTX3) derived from small and medium antral follicles before and after oocyte maturation. Methods: Only the cumulus oocyte-complexes (COCs) derived from small antral follicles were induced with [Pre-SF(+)hCG] or without [Pre-SF(-)hCG] the addition of human chorionic gonadotropin (hCG) during the last 7 h of the pre-IVM period before undergoing the regular culture system. The mature oocytes were investigated on embryonic development after parthenogenetic activation (PA). Melatonin (10^-7 M) was supplemented during in vitro culture (IVC) to improve the developmental potential of these porcine pathenotes. Results: A pre-IVM culture system with hCG added during the last 7 h of the pre-IVM period [Pre-SF(+)hCG] effectively supported small antral follicle-derived oocytes and increased their nuclear maturation rate. The oocytes derived from medium antral follicles exhibited the highest nuclear maturation rate in a regular culture system. Compared with oocytes cultured in a regular culture system, those cultured in the pre-IVM culture system exhibited considerable overexpression of HAS2, PTGS2, and TNFAIP6. Porcine embryos treated with melatonin during IVC exhibited markedly improved quality and developmental competence after PA. Notably, melatonin supplementation during the IVM period can reduce and increase the levels of intracellular reactive oxygen species (ROS) and glutathione (GSH), respectively. Conclusion: Our findings indicate that the Pre-SF(+)hCG culture system increases the nuclear maturation rate of small antral follicle-derived oocytes and the expression of genes involved in cumulus expansion. Melatonin supplementation during IVC may improve the quality and increase the blastocyst formation rate of porcine embryos. In addition, it can reduce and increase the levels of ROS and GSH, respectively, in mature oocytes, thus affecting subsequent embryos.

• Proceedings of the KSAR Conference
• /
• 2004.06a
• /
• pp.243-243
• /
• 2004

Purpose: This study was conducted to obtain the relationship between initial size of pre-antral follicles (PAF) and intra-follicular oocytes (IFO) and their in vitro growth (IVG) in medium without gonadotropins (Gns) using PAF isolated from mouse ovaries mechanically. (omitted)

• Proceedings of the Korean Society of Embryo Transfer Conference
• /
• 2004.10a
• /
• pp.67-67
• /
• 2004

• 대한생식의학회:학술대회논문집
• /
• 2005.06a
• /
• pp.109.2-110
• /
• 2005

• Reproductive and Developmental Biology
• /
• v.32 no.2
• /
• pp.111-115
• /
• 2008

The aim of this study was to examine more effective cryoprotectant for the cryopreservation of mouse preantral follicles. Enzymetically isolated preantral follicles from 12-day-old mice were cryopreserved by a slow freezing protocol with 1.5 M propanediol (PROH), dimethyl sulphoxide (DMSO) or glycerol (GLY) and then grown and matured in vitro for 11 days after thawing. The survival of preantral follicles immediately after freezing and thawing was not different among the PROH (68.2%), DMSO (72.4%) and GLY (72.1%). After grown and matured in vitro, the rates of survival and metaphase II oocytes were 54.9% and 36.6% for PROH which was significantly higher rates (p<0.05) compared with the rates obtained from DMSO (16.9% and 9.0%) and GLY (16.3% and 7.5%). The diameter of metaphase II oocytes from pre antral follicles frozen in PROH ($67.4{\pm}1.8\;{\mu}m$) was significantly (p<0.05) smaller than that of the fresh preantral follicles ($69.1{\pm}2.3\;{\mu}m$). The results from the present study revealed that PROH is more suitable cryoprotectant for the cryopreservation of mouse preantral follicles.

• Korean Journal of Animal Reproduction
• /
• v.24 no.4
• /
• pp.347-356
• /
• 2000

포유동물의 난소 내에는 많은 수의 primordial follicles 과 preantral follicles 이 존재하고 있으며, 이것은 수정란을 체외생산하기 위한 잠재적인 난자의 공급원이 될 수 있다. 생쥐 preantral follicles 내에 존재하는 난자의 체외성장과 발달을 위하여 몇몇 배양체계가 개발이 되었으며, 적당한 배양조건에서 감수분열 능력이 없는 preantral follicles 내의 난자가 체외배양을 통하여 난자 직경이 증가하고 완전한 핵성숙을 하는 것으로 나타났다. 또한, 체외성장 및 성숙된 난자로부터 생쥐 산자의 성공적인 생산은 preantral follicles 내의 난자가 체외배양을 통해서도 완전한 발달능력을 얻을 수 있음을 입증하였다. 그렇지만, 생쥐 preantral follicle로부터 수정란의 체외생산능력은 매우 낮은 것으로 보고되고 있다. 한편 사람을 비롯한 돼지, 소와 같은 중ㆍ대가축의 경우에는 pre antral follicle의 체외배양을 통하여 감수분열능이 있는 단계의 난자로까지의 발달이 아직 보고가 되지 않고 있다. 따라서 지금까지의 연구결과들을 종합해 볼 때, preantral follicles의 체외배양조건을 개선하거나 새로운 배양체계를 개발에 대한 많은 연구가 요구되고 있다. 사람과 중ㆍ대가축의 preantral follicles의 체외배양체계의 확립은 우수한 형질을 가진 동물의 확장, 희귀동물 혹은 멸종위기 동물의 보존에 이용될 수 있을 것이며, 그리고 암치료를 위하여 화학적, 방사선 치료를 받아야 하는 여성에게서 향후에 불입치료를 위한 방법으로 활용될 수 있을 것으로 기대된다.

• Asian-Australasian Journal of Animal Sciences
• /
• v.17 no.12
• /
• pp.1647-1651
• /
• 2004

The follicles (1.8 to 7.8 mm in diameter) were recovered from the ovaries in marketed pigs and the number of granulosa cells, the diameter of oocytes obtained from different development stages of the follicles and follicular fluid levels were determined. Correlations between size measurements and cell counts as well as the diameter of antral follicles and oocytes were also investigated. The results indicated that, while expanding in size, follicle numbers decreased with a greater atretic proportion. Granulosa cells increased in numbers continuously and remained unchanged beyond the size of 200 ${mm}^3$ in non-atretic follicles, whereas a sudden drop of granulosa counts was observed in atretic follicles. Follicular fluid, on the other hand, linearly increased its volume with follicle size and differed little between those of non-atretic and atretic follicles. Diameters of oocytes in non-atretic follicles increased to its maximum when follicles expanded to 150 ${mm}^3$ and maintained its size during later follicular expansion. It is concluded that, for in vitro culture, the optimal size of porcine follicle should be between 150 to 180 ${mm}^3$if they are collected from pre-pubertal gilts of marketing size slaughtered in an abattoir.

• Asian-Australasian Journal of Animal Sciences
• /
• v.16 no.7
• /
• pp.1071-1086
• /
• 2003

Reproductive biotechnologies continue to be developed for genetic improvement of both river and swamp buffalo. Although artificial insemination using frozen semen emerged some decades back, there are still considerable limitations. The major problem appears to be the lack of efficient methods for estrus detection and timely insemination. Controlled breeding experiments in the buffalo had been limited and similar to those applied in cattle. Studies on multiple ovulation and embryo transfer are essentially a replica of those in cattle, however with inherent problems such as lower number of primordial follicles on the buffalo ovary, poor fertility and seasonality of reproduction, lower population of antral follicles at all stages of the estrous cycle, poor endocrine status and a high incidence of deep atresia in ovarian follicles, the response in terms of transferable embryo recovery has remained low with 0.51 to 3.0 per donor and pregnancy rates between 15 to 30%. In vitro production of buffalo embryos is a valid alternative to recovery of embryos by superovulation. This aspect received considerable attention during the past decade, however the proportion of embryos that develops to the blastocyst stage is still around 25-30% and hence the in vitro culture procedures need substantial improvement. Embryo cryopreservation procedures for direct transfer post thaw need to be developed for bubaline embryos. Nuclear transfer and embryo cloning is a technique that has received attention in various species during recent years and can be of immense value in buffaloes as they have a low rate of embryo recoveries by both in vitro and in vivo procedures. Gender pre-selection, genome analysis, gene mapping and gene transfer are a few of the techniques that have been studied to a limited extent during recent years and are likely to be included in future studies on buffaloes. Very recently, reproductive biotechnologies have been applied to feral buffaloes as well, but the results obtained so far are modest. When fully exploited they can play an important role in the preservation of endangered species.