Preservasi ovarium, isolasi dan kultur folikel in vitro pada domba

Abstract

Ovine ovary contains about 36.000 of preantral follicles of which only 0.01% ovulates in the reproductive life. In the last decades, many studies have been carried out focusing on preservation, isolation and culture of preantral follicles from several species ovaries. In this study the effect of cooling and freezing of ovine ovarian tissue were examined on follicles morphology, number and quality of the follicles. The follicles were isolated by different methods. The developmental competence of follicles cultured in vitro were evaluated postpreservation. The study was carried out in 2 experiments. Experiment I, ovaries were maintained in PBS at -20 oC and room temperature (RT) for 24 h, and at 5 oC for 24 h and 72 h, and vitrification by 10, 20, 30 min equilibration time. After preservation and warming, the tissues were prepared for histological examination. Experiment 2, follicles isolated from fresh ovaries both mechanically and enzymatically. Then, preantral follicles were isolated from a) fresh ovaries (control), ovaries were stored at 5 oC for: b) 24 h , c) 48 h, d) 72 h, and vitrified cortex tissue (after 6 d in vitro culture). Preantral follicles (220-240 μm) were cultured in αMEM supplemented with 5% FCS, 100 mIU/ml r-FSH and ITS (consist of 5μg/ml insulin, 5 μg/ml transferin, 5 ng/ml selenium) up to ovulation stage. No follicle survived after 24 h storage at RT. The percentage of morphologically normal follicles was significantly reduced in ovarian tissue stored at -20 oC for 24 h and at 5 oC for 24 h and 72 h,5 oC for 24 h gave the better results. The antral follicles were damaged in all treatment. Exposing tissue to equilibration medium for 10 min had higher morphologically normal of cooledwarmed follicles, but had fewer morphologically normal follicles than fresh ovary. Experiment 2 shown that enzymatic method yielded more follicles than mechanic method, but fewer intact follicles. Follicle development up to ovulation of 5 oC storage for 24 h was equal to fresh follicles. Ovarium preserved at 5 oC for 48 h resulted in a less number of follicles that reach ovulation stage than others.Vitrification slighty reduced developmental competence in vitro. We conclude that storage of ovine ovaries for up to 24 h at -20 oC, RT, and 5 oC declined the number of morphologically normal follicles, 5 oC storage gave the better results. Primordial follicles preserved their morphology intactness better than growing follicles. Good morphology of follicles was confirmed when exposing tissue to equilibration medium for 10 min before freezing. Ovary could be storage at 5 oC up to 48 h to maintain follicles viability. Vitrification slighty reduced follicle developmental competence in vitro.

Folikel merupakan unit struktural dan fungsional dasar dari ovarium mamalia yang menyediakan lingkungan mikro yang diperlukan untuk pertumbuhan dan maturasi oosit. Ovarium domba mengandung sekitar 36.ooo folikel preantral, diantaranya hanya 0,01% yang ovulasi sepanjang masa produktifnya. Dalam dekade terakhir, banyak penelitian telah dilakukan dengan fokus preservasi, isolasi, dan kultur folikel dari berbagai spesies. Pada penelitian ini, dipelajari pengaruh pendinginan dan pembekuan jaringan ovarium terhadap morfologi, jumlah dan kualitas folikel domba yang diisolasi dengan metode berbeda serta kompetensi perkembangan folikel in vitro pasca preservasi. Penelitian ini dilaksanakan dalam 2 tahap. Pada tahap 1 dilakukan 2 eksperimen preservasi ovarium. Eksperimen 1, ovarium disimpan dalam larutan PBS pada suhu -20 oC dan suhu kamar selama 24 jam, dan suhu 5 oC selama 24 jam dan 72 jam. Setelah penyimpanan, folikel-folikel dievaluasi secara histologis. Dalam Eksperiment 2, cortex ovarium dipisahkan dari ovarium dan dibentuk dalam potongan berukuran ±1 mm3. Potongan jaringan diletakkan di atas hemistraw dan ditransfer ke larutan ekuilibrasi masing-masing selama 10, 20, dan 30 menit pada suhu kamar, selanjutnya dipindahkan ke larutan vitrifikasi selama 3 menit. Hemistraw beserta jaringan dicelupkan dalam nitrogen cair. Setelah penghangatan, jaringan dievaluasi secara histologis. Pada tahap kedua dilakukan isolasi dan kultur in vitro folikel. Eksperimen 3, potongan cortex ovarium berukuran ± 1 mm3 diinkubasi dalam collagenase 1 mg/ml (C1) dan collagenase 2 mg/ml (C2) masing-masing selama 15, 30, 45, dan 60 menit. Folikel-folikel dari cacahan jaringan cortex juga diisolasi secara mekanik (M) menggunakan jarum 26G. Hasil isolasi dari ketiga perlakuan diamati dibawah mikroskop menggunakan pembesaran obyektif 40 kali. Eksperimen 4, folikel-folikel preantral diisolasi pada ovarium segar (kontrol), ovarium yang disimpan pada suhu 5 oC selama 24 jam (T5-24), 48 jam (T5-48), dan 72 jam (T5- 72) dan jaringan cortex hasil vitrifikasi (V). Folikel-folikel berukuran 220-240 μm dikultur dalam medium αMEM disuplementasi 5% FCS, 100 mIU/ml r-FSH dan ITS (terdiri dari 5μg/ml insulin, 5 μg/ml transferin, 5 ng/ml selenium) sampai ovulasi.