• 농업과학연구
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• 제48권1호
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• pp.163-170
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• 2021

Bacterial infections in the female reproductive tract negatively affect ovarian function, follicular development, and embryo development, leading to the eventual failure of fertilization. Moreover, bacterial lipopolysaccharides (LPS) can interfere with the immune system and reproductive system of the host animal. Therefore, this study examined the effect of LPS on the in vitro maturation (IVM) of pig oocytes. Oocytes were matured in TCM199 medium in the presence of varying concentrations of LPS (0 - 50 ㎍·mL^-1). The maturation rate, cortical granules (CGs) migration, and chromosome alignment were subsequently evaluated during the meiotic development of the oocytes. We observed a dose-dependent and significant decrease in the metaphase II (MII) rate with increasing concentrations of LPS (97.6% control [0 ㎍·mL^-1 LPS] vs. 10.4-74.9% LPS [1 - 50 ㎍·mL^-1], p < 0.05). In addition, compared to the control oocytes without LPS, higher levels of abnormal CGs distribution (18.1 - 50.0% LPS vs. 0% control), chromosome/spindle alignment (20.3 - 56.7% LPS vs. 0% control), and intracellular ROS generation were observed in oocytes matured with LPS (p < 0.05). Nitrite levels were also increased in the maturation medium derived from the oocytes matured with LPS (p < 0.05). These results indicate that LPS induces oxidative stress during IVM and affects oocyte maturation, including CGs migration and chromosome alignment of pig oocytes.

• Animal Bioscience
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• 제37권6호
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• pp.1007-1020
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• 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.

• Reproductive and Developmental Biology
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• 제29권4호
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• pp.223-227
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• 2005

In vitro maturation of porcine immature cumulus-enclosed oocytes can be enhanced by co-incubation with spermatozoa even before fertilization. The aim of this study was to determine whether the addition of spermatozoa into the culture medium can stimulate the meiosis resumption of porcine cumulus-enclosed oocytes arrested at germinal vesicle (GV). Cumulus-enclosed oocytes (CEOs) were collected from follicles of 3 to 5mm diameter. Porcine CEOs were cultured in tissue culture medium containing various meiosis inhibitors and spermatozoa. Oocytes were examined for evidence of GV and GV breakdown after 24 h culture. After 24 h culture $43.8\%$ of oocytes cultured in only TCM 199 remained at GV stage whereas $56.2\%$ of oocytes were able to resume meiosis. When porcine CEOs were cultured in the medium with meiosis inhibitor such as, dibutyryl cAMP (dbcAMP) and forskolin (Fo), more than $90\%$ of oocytes were not able to resume meiosis. However, co-culture of porcine CEOs with spermatozoa was able to overcome the inhibitory effect of dbcAMP and Fo. Irrespective of the presence of 3-isobutyl-1-methylxanthine (IBMX), no difference was observed in the proportion of oocyte reached germinal vesicle breakdown (GVBD). The present study suggests that dbcAMP and Fo prevent the spontaneous maturation of competent oocyte in culture after isolation from follicles and that mammalian spermatozoa contain a substance(s) that improves meiosis resumption in vitro of porcine cumulus-enclosed oocytes.

• Clinical and Experimental Reproductive Medicine
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• 제49권3호
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• pp.175-184
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• 2022

Objective: The aim of this study was to evaluate the impacts of platelet-rich plasma (PRP) and conditioned medium (CM) derived from endometrial stromal cells on mouse preantral follicle culture in a two-dimensional system to produce competent mature oocytes for fertilization. Methods: In total, 240 preantral follicles were isolated from female mouse ovarian tissue and divided into four groups. The preantral follicles were isolated three times for each group and then cultured, respectively, in the presence of alpha minimum essential medium (control), PRP, CM, and PRP+CM. The in vitro growth, in vitro maturation, and cleavage percentage of the preantral follicles were investigated. Immunocytochemistry (IHC) was also conducted to monitor the meiotic progression of the oocytes. Additionally, the mRNA expression levels of the two folliculogenesis-related genes (Gdf9 and Bmp15) and two apoptosis-related genes (Bcl2 and Bax) were investigated using real-time polymerase chain reaction. Results: In the PRP, CM, and PRP+CM groups, the preantral follicle maturation (evaluated by identifying polar bodies) were greater than the control group. The cleavage rate in the CM, and PRP+CM groups were also greater than the control group. IHC analysis demonstrated that in each treatment group, meiotic spindle was normal. In the PRP+CM group, the gene expression levels of Bmp15, Gdf9, and Bcl2 were greater than in the other groups. The Bax gene was more strongly expressed in the PRP and control groups than in the other groups. Conclusion: Overall, the present study suggests that the combination of CM and PRP can effectively increase the growth and cleavage rate of mouse preantral follicles in vitro.

• Animal cells and systems
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• 제5권1호
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• pp.51-57
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• 2001

We have previously shown that oocyte maturation is induced by an immobilized progesterone, progesterone-3-carboxymethyloxime - bovine serum albumin conjugate (P-BSA) in Rana dybowskii. In this study, we confirmed the maturation inducing activity of P-BSA on Xenopus oocyte and examined the binding character of the immobilized progesterone on the surface of Xenopus oocytes after removal of the vitelline layer. P-BSA induced maturation of Xenopus oocytes but E-BSA failed to do so as observed in Rana. Binding of the immobilized progesterone, fluorescein isothiocyanate-labeled progesterone-3-0-carboxymethyloxime-BSA (P-BSA-FITC) on the devitellined oocytes surface was examined by fluorescence confocal microscopy. The binding affinity of P-BSA-FITC to the devitellined oocyte was higher than that of estrogen-BSA-FITC (E-BSA-FITC) or testosterone-BSA-FITC (T-BSA-FITC). The binding disappeared in the presence of excess free progesterone but not in the presence of free estrogen. Maximum binding occurred after two-hours of incubation with P-BSA-FITC at pH 7.5. Stronger binding occurred in oocytes at stage Vl than stage IV, and in vitro treatment of hCG enhanced the binding. Taken together, these results suggest that a specific receptor for progesterone exists on the plasma membrane of Xenopus oocytes and that progesterone acts initially on this putative receptors and triggers generation of membrane-mediated second messengers during the early stage of oocyte maturation In amphibians.

• 한국가축번식학회지
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• 제17권2호
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• pp.75-83
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• 1993

Purine has been identified in the preparation of follicular fluid and shown an activity in maintaining oocyte meiotic arrest. Therefore this study was performed to examine the inhibitory effect of purine on germinal vesicle break down(GVBD) in the presence and absence of human fetal cord serum(HFCS) or human mature follicular fluid(HMFF), as a protein source, in vitro culture. Immature oocytes(GV stage) were collected from ovaries of 21∼28 days old ICR mice by puncturing the antral follicles with a fine needle, at 48 hrs after PMSG injection. Some of the oocytes were denuded by drawing the cumulus-enclosed(complex) oocytes in and out of a pasteur pipet. Complex oocytes and denuded oocytes were cultured 3 hrs. in T6 media containing 0.75mM adenosine or/and 4mM hypoxanthine, with HFCS or HMFF. Their GVBD rates were observed at every 1 hr. during the culture time. Both adenosine and hypoxanthine have shown a time-dependent inhibitory effect on GVBD in complex and denuded oocytes and the inhibitory effect was maximized in culture medium containing hypoxanthine and adenosine. HFCS and HMFF increased the GVBD rates in the presence of the purines, thus HFCS and HMFF may contain a factor that could reverse the inhibitory effect of purines. Also complex oocytes were more sensitive to not only the inhibitory effect of purines but the promoting action of HMFF on GVBD than denuded oocytes. Therefore it was reconfirmed that granulosa cells play an important part in meiotic arrest and resumption.

• 한국가축번식학회지
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• 제10권2호
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• pp.211-217
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• 1986

This experiment was conducted to determine the effect of FSH on in vitro maturation and in vitro fertilizing ability of oocytes recovered from normal follicles of different sizes in superovulated rabbits. Follicular oocytes recovered were cultured in modified Ham's F12 medium containing 0, 0.1, 1.0 and 10$\mu\textrm{g}$ FSH/ml for 18 hours and investigated the degree of cumulus cells expansion and nuclear maturation, which were fertilized with in vivo capacitated rabbit sperm. 1. The number of normal follicles<1.5mm, 1.6 to 2.5mm and> 2.5mm in diameter at 16 to 18hrs after HCG administration was 4.8 (38.8%), 5.5(45.4%) and 3.3(15.8%), respectively. Average percent of oocytes recovered was 69.7% and larger follicles tended to have a higher percent, recovery rate than smaller follicles. 2. The degree of cumulus expansion in medium containing 0.1$\mu\textrm{g}$ FSH/ml was similar to that of control, but markedly decreased under the level of above 1$\mu\textrm{g}$ FSH/ml. The proportions of oocytes which reached the second meiotic metaphase were 57.1, 61.5, 43.8 and 45.0% in medium containing 0, 0.1, 1.0 and 10$\mu\textrm{g}$ FSH/ml, respectively. Oocytes from larger follicles showed a higher nuclear maturation than that from smaller follicles. 3. In vitro fertilization rate of oocytes matured under 1$\mu\textrm{g}$ FSH/ml was slightly, not significant, higher than that of others. 4. Progesterone level in follicular fluid was about 67 to 71ng/ml with no difference in follicular sizes and estradiol-17$\beta$ level was under 25pg/ml.

• Clinical and Experimental Reproductive Medicine
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• 제28권1호
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• pp.1-12
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• 2001

Objective: The present study was done to clarify the effects of nicotine and nicotine tartrate on the mouse oocyte maturation in vitro. Methods: GV (germinal vesicle) oocytes were isolated from Graafian follicle of ovaries with sharp needles under a stereomicroscope from female mouse of ICR strain (4 weeks old). Collected oocytes were cultured for 17 hours at $37^{\circ}C$, 5% $CO_2$ in air and 100% humidified condition in incubator. New MHBS was the basic medium used in which nicotine, nicotine tartrate, and mecamylamine (antagonist of nicotinic acetylcholine receptor) were added depending on the experimental group. GV oocytes were cultured in one of these media. Results: Nicotine ($300{\mu}M{\sim}5mM$) had no effects on GVBD (germinal vesicle breakdown) compared to the control, but increasing concentration of nicotine led to an decrease in the first polar body formation. However, nicotine ($10{\sim}500{\mu}M$) induced GVBD in a dose-dependent manner of GV oocytes in a medium containing dbcAMP. Nicotine tartrate ($50{\mu}M{\sim}5mM$) had no effects on GVBD compared to the control but, increasing concentration of nicotine tartrate led to an decrease in the first polar body formation. Mecamylamine $10{\mu}M$ added to the medium containing nicotine ($300{\mu}M{\sim}5mM$) showed higher percentage of the first polar body formation compared to the nicotine ($300{\mu}M{\sim}5mM$) treatment group. Mecamylamine $10{\mu}M$ added to the medium containing nicotine tartrate ($50{\mu}M{\sim}5mM$) showed higher percentage of the first polar body formation compared to the nicotine tartrate ($50{\mu}M{\sim}5mM$) treatment group. Conclusion: The present study suggest that nicotine and nicotine tartrate have the harmful effects on the meiotic maturation of the mouse oocytes in vitro. However, mecamylamine block harmful effects of nicotine and nictine tartrate.

• Clinical and Experimental Reproductive Medicine
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• 제8권1호
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• pp.1-12
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• 1981

면역학적인 방법을 사용하여 가돈 여포액내의 특이단백질의 존재여부와 이들을 추출하여 난자성숙에 미치는 영향을 조사한 결과 다음과 같았다. 1. 가돈 혈청 및 혈장에는 존재하지 않는 면역학적으로 특이한 두 단백질이 가돈 여포액내에서 검출되었다. 2. 이들 두 단백질은 전기영동적으로 각각 fast alpha-I globulin 과 beta-globulin 의 이동성을 나타냈다. 3. 이들 두 단백질은 생쥐 여포난자의 성숙을 억제시켰다. 본 실험의 결과로 미루어 가돈 여포액내에는 난자성숙을 억제시키는 특이단백질이 존재하는 것으로 사료된다.

• 한국수정란이식학회지
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• 제20권1호
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• pp.63-69
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• 2005

본 연구는 개의 미성숙난자의 낮은 성숙율을 개선하기 위하여 자연발정온 개의 생식기관에 미성숙 난자를 이식하여 체내 배양 난자를 회수하여 회수된 난자의 핵 성숙율을 조사한 결과는 다음과 같다. 1. 미성숙난자를 24, 48 및 72시간 동안 체외성숙을 유도하였을 때 성숙시간에 따른 체외성숙율에는 유의적 차이는 인정되지 않았다. 2. 체내 배양 기간에 따른 난자의 회수율은 배양기간이 길어질수록 유의적으로 낮은 회수율을 보였으며, 배양기간이 증가할수록 난자의 생존율은 유의적으로 (P<0.05) 저하되었고 사멸된 난자의 비율도 또한 증가하였다. 3. 회수된 난자는 4, 5, 6일간의 체내 배양기간에 관계없이 대부분 GV상태로 (20/34; 6/15; 4/7)존재하였으며, 성숙된 난자는 체내 배양 4일째 회수된 난자에서 $5.8\%(2/34)$의 난자가 M II까지 발달한 것으로 조사되어 개의 미성숙난자의 체내 배양은 개 난자의 핵성숙을 개선하지 못하였다. 이상의 결과에서와 같이 개의 미성숙난자의 체내 배양은 M II 단계까지의 발달에 기여하지 못한 것으로 판단되며, 이는 개 미성숙난자의 핵발달은 난포에서의 자극과 활성화가 요구되는 것으로 판단된다.