Although assisted reproductive technology is very useful to develop novel and therapeutic biomaterials for reproduction, research on molecular mechanism of folliculogenesis in pig is not clear. Therefore, the alteration of gene expression during follicular development in pigs was examined in this study. The expression of folliculogenesis-related genes was quantified in preantral (250～300 μm) and antral (>300 μm in diameter) follicles, and overall gene expression was evaluated by a genome-wide microarray. The microarray results showed that 219 genes were differentially expressed, and of those, 10 and 22 known genes showed higher and less expression at the preantral stage than at antral stages, respectively. Among them, the expression of NR0B1, PPARG, GATA4, and ANXA2 genes related to folliculogenesis was validated by quantitative real-time PCR analysis. The expression of PPARG and GATA4 genes were increased at antral stages, but a significantly stage-specific increase (p<0.05) was only detected in annexin A2 (ANXA2) in antral-stage follicles. The expression of NR0B1 genes was increased at preantral stage and these patterns of gene expression were comparable to the results obtained by microarray analysis. We propose that the systematical regulation of genes supporting specific follicle stage should be employed for improved in-vitro folliculognesis.

This study was conducted to analyze the expression pattern of inhibitor of DNA binding proteins (Id)1 and Id2 mRNA on folliculogenesis in rat ovary. The ovaries were obtained from 27 days old Sprague-Dawley rat, fixed, dehydrated, and paraffin embedded. For in situ hybridization, anti-sense and sense Idl and Id2 cRNA probes were prepared and applied to the ovarian section. The ovarian sections were coated with NTB-2 emulsion. After that, the slides were developed and counterstained with hematoxylin and eosin staining. In oocytes, the hybridizational signals of Id1 mRNA were strong in primordial and primary follicles, however, there were no signals in that of atretic or preovulatory follicles. The Id2 mRNA signals were also strong in the oocytes of primordial, primary and secondary follicles. Interestingly, the Id2 mRNA was expressed specifically granulosa cells, but nor in oocyte or theca cells in dominant and preovulatory follicles. Based on these results, Id1 and Id2 mRNA was expressed specifically at follicle stages and follicular tissue and might be closely related with follicle development.

DNA 결합 단백질 억제(Inhibitor of DNA binding protein) 또는 분화억제(Inhibitor of differentiation) 인자인 Id는 네 종류(Id1-4)가 있으며, 세포의 증식과 분화, 혈관 형성 및 세포자가사멸 등에 정 또는 부의 조절인자로서 널리 알려져 있다. 하지만 아직까지 번식생리 분야에서 이들 유전자들의 발현이나 기능에 관해 알려진 것은 거의 없다. 따라서 본 연구에서는 쥐 난소에서 난포의 발달에 따른 Id3 mRNA의 발현 양상을 살펴보고자 실시하였다. 쥐에게 PMSG 주사 후, 3, 6, 12, 24, 36 및 48시간째에 난소를 회수하여 고정, 탈수 및 파라핀 포매를 실시하였다. In situ hybridization 실험을 위하여 anti-sense와 sense Id3 cRNA 탐침자를 제작한 다음 난소 절편에 반응시켰다. 반응이 끝난 난소 절편은 NTB-2 유광제에 노출시킨 후 현상액에 반응시켰다. 모든 처리가 끝난 슬라이드는 H&E 염색을 실시한 다음 현미경하에서 hybridization 감도를 1+에서 4+로 구분하여 평가하였다. 난자에서는 Id3 mRNA 감도가 원시와 제1차 난포에서 ≥2+으로 확인되었으나, 제2차, 우성 및 배란전 난포에서는 1+ 이하로 관찰되었다. 한편, 과립막세포에서는 우성과 배란 전 난포에서 Id3 mRNA가 3+ 또는 4+로 강하게 발현되는 것을 확인하였다. 이상의 결과를 종합하여 보면, Id3 mRNA는 난포의 발단 단계 또는 난포세포에 따라 특이적으로 발현하는 것으로 보아 난포의 발달과 밀접한 연관이 있을 것으로 사료된다.

Sulfotransferase 1E1 (SULT1E1, EST) is responsible for the sulfation and inactivation of betaestradiol at physiological concentrations. SULT1E1 null mice have reduced fertility by the disfunction of placenta and ovulation. Based our previous data it was revealed that the ovulation ability of 6-month old tiEsr1KO female mice is similar with the SULT1E1 female mice. In this study the possible relations in ovulation between estrogen and SULT1E1 was examined using real-time PCR and RIA methods in tiEsr1KO model mice. During the induction of superovualtion SULT1E1 gene expression peaked just before ovulation (6 hr posthCG injection) in control mice. As expected the expression patterns were similar between control and 4-weelk old tiEsr1KO female mice. Serum levels of E2 were increased in both tiEsr1KO mice and wild type mice but its level was higher 3 times in tiEsrKO mice than the wild type. Its levels became same between them after hCG administration. On the other hand, 6-month old mice shows the the dramatical increasing the SULT 1E1 expression during folliculogenesis, their expression was increased more than 100 times after hCG 6hr compared with PMSG 12hr control. In contrast with the 4-week old tiEsr1KO female mice, the expression levels of SULT1E1 gene expression were higher levels more than 20 times at 12 hr post PMSG injection. On the other hand, the expression levels of the other SULT family genes, SULT1A1 and SULT2A1 were very low compared with the SULT1E1 and did not show fluctuation during that period. Based on these results, it is suggested that the functional roles of estrogen during folliculogenesis may be regulated by SULT1E1through the metabolism of 17-beta estradiol and that the expression of SULT1E1 gene may be under the control of the levels of estrogen.

Insulin like3 (INSL3, Relaxin like factor) is a critical regulator in testis translocation through Leucin rich G-protein coupled receptor 8 (Rxfp2,LGR8) during embryogenesis. In female, INSL3 and their receptor expressed in growing follicle and revealed their function in oogenesis. However, its role is not much evaluated. 6 weeks old C57BL/6 female mice used for measure the expression of INSL3 mRNA and their receptor expression by follicular stage. Follicle cell specific RNA were got from the theca cell and granulose cells which were isolated using LCM. To know the role of INSL 3 in theca cell, Rxpf2 were overexpressed during primary culture of theca cells, which were isolated from tertiary follicles after 12 hr PMSG injection and transduced the Rxfp2 using lenti virus. After 10 days of culture, the proliferation of theca cells was analyzed using EdU. Using Alzet osmotic pumps INSL3 was administered for 3 days into the ovary during superovulation induction. The INSL3 mRNA levels were significantly high in the theca cells of preovulator follicles after hCG injection. But granulose cells showed decreasing expression by growth of follicle. INSL3 stimulated the proliferation of theca cells in vitro which overexpressed the Rxpf2. By the administration of INSL3 into ovary caused the dramatical decrease the number of ovulated oocyte. Based on these results, we know that INSL3 stimulates the theca cell proliferation in as follicle stage specific manners, and estrogen is a modulator of this INSL3 mRNA expression. It suggests that the disturbance of the expression regulation of INSL3 and its receptor cause the unregulated theca cell proliferation and failing the rupture of grown follicle.