IDENTIFIKASI DAN UJI BIOAKTIVITAS GROWTH FACTOR DAN
HORMON STEROID SEKS HASIL BIAKAN MONOLAYER SEL
HEPAR DAN SEL KUMULUS SAPI
MULYATI, SRI
Promotor : Prof. Dr. Laba Mahaputra, drh., M.Sc.
ESTROGEN; PROGESTERONE; LIVER CELL
KKA KK Dis K 31/12 Mul i
Copyright© 2011 by Airlangga University Library Surabaya
ABSTRACT
IDENTIFICATION AND BIOACTIVITY TEST OF GROWTH FACTOR AND SEX STEROID HORMONE PRODUCED BY BOVINE LIVER AND CUMULUS
CELL MONOLAYER CULTURE
Cumulus cells and liver cells (hepatocyte) have an ability to produce IGF-1 and estrogen through monolayer culture, but the concentration and the bioactivity in embryo was not clear.The aim of this study was to monitor, analysis and identify bioactivity of IGF-1 and estrogen there found in product cell. Liver cell culture of cattle was prepared by grinding liver tissue followed by trypsination and repeatedly centrifuged were obtained hepatocyte. Cumulus cells were obtained from aspirating ovarian follicles with diameter 2-6 mm. Liver and cumulus cells were cultured in TCM 199 + FCS 10% + BSA 10%. Cell concentration was 1.9 x 106 /ml medium then culture was incubated in incubator at 38.5oC 5% CO2 for 3,6,9,12 days. IGF-1 concentration media after culture was measured by IRMA, while estrogen and progesterone using RIA technique. To minimize progesterone concentration in product cell it was used binding technique using antiprogesterone (antiP4) coated polyesterene tubes. Bioactivity test of IGF-1 and estrogen obtained from this research as supplementation media in in vitro fertilization and as embryo culture media was performed at 2 and 6 days after fertilization by observating embryo cleavage 2-8 cells and embryo development up to morula stage. The mitogenic and antiapoptotic of IGF-1 and estrogen were examined by the reduction of apoptosis in embryo resulted. The result of research showed that 6 days incubation period resulted the most confluent monolayer compared to the other incubation period, either in liver or cumulus cells, therefore resulting the highest concentration of IGF-1 as well as estrogen. In liver cell culture IGF-1 concentration was higher compared to these of the cumulus cells, meanwhile in cumulus cells the estrogen was found higher than IGF-1. AntiP4 could be used to absorb progesterone resulted from monolayer culture. IGF-1 and estrogen from monolayer culture before and after progesterone absorbtion resulted in cleavage rate were 27.48% and 53.61%, respectively stage morula embryo rate were 5.73% and 27.59% , and could also reduce the incidence apoptosis in the embryos cultured in liver cell were 23.71% and in cumulus cells were 31.01%, respectively while without supplemented apoptotic rate in embryo cell was 70.58%. The conclusion of this research was that monolayer culture of bovine liver and cumulus cells could produce IGF-1 and estrogen growth factor which acted as mitogenic substances that could be used
as supplemen media in in vitro fertilization and embryo development as well as able to reduce the incidence of apoptosis in embryo resulted.
Keywords : IGF-1, estrogen, progesterone, monolayer culture, liver cell (hepatocyte), cumulus cell.
SUMMARY
IDENTIFICATION AND BIOACTIVITY TEST OF GROWTH FACTOR AND SEX STEROID HORMONE PRODUCED BY BOVINE LIVER AND CUMULUS
CELL MONOLAYER CULTURE
The implementation of biotechnology is an attemp for increasing the efficiency of livestock reproduction particularly to obtain good quality and quantity livestock. Embryo transfer is one technique considered efficient and effective in the area of reproduction. The success of this technology requires quality embryos. In vivo embryo production is limited by the ability of donor females in producing embryo. For this case, in vitro embryo production is an alternative source of embryo.
Growth factors i.e. Insulin-Like Growth Factor-I (IGF-I) and estrogen are two mitogenic substances that have been proved can be used to stimulate and accelerate cell growth.
IGF-I or somatomedin is a polypeptide growth factor secreted by liver and a various tissue in response to growth hormone stimulation. IGF-I contains 70 amino acids with a molecular weight of 7649 Dalton or 7.65 kDa. Estrogen is a steroid hormone especially produced by growing follicles in the ovary, corpora lutea and placenta with a molecular weight of 300–400 Dalton (272.3 Dalton). IGF-I and estrogen through paracrine, autocrine and endocrine manners stimulate mitogenic activities in proliferating cells.
Liver and ovary are organs that can produce IGF-I and estrogen. Liver is the main IGF-I producer while ovary is the main producers of sex steroid estrogen and both of them can be developed through monolayer cell culture with the supplementation of Foetal Calf Serum (FCS) or Bovine Serum Albumine (BSA) as precursor substances at monolayer cultures used.
From the descriptions, research has been conducted purposively to quantify the concentration of IGF-I and steroid sex hormone estrogen and progesterone in the media of liver and cumulus cell monolayer culture that used liver and ovaries of cattle slaughtered in abattoir.
This research was conducted in stages. The first stage was the production and harvest of liver and cumulus cell monolayer culture. Liver culture was prepared by grinding liver tissue trypsination and repeated centrifugation until liver cells (hepatocytes) were obtained. Cumulus cells were obtained from aspirating follicles with diameter 2-6 mm. Then liver and cumulus cells were cultured separately in TCM 199 + FCS 10 % + BSA 10 %. Cell culture concentration was 1.9 x 106/ml medium then was incubated in an incubator at 38.5 oC, 5 % CO2 in air for 3, 6, 9 and 12 days. The second stage was the quantification of IGF-I concentration in the media of liver and cumulus cell monolayer culture measured by Immuno Radio Metric Assay (IRMA), estrogen and progesterone measured with Radio Immuno Assay (RIA). The third stage was the separation or absorbtion of progesterone that is an anti-mitogenic substance from IGF-I and estrogen that were mitogenic substances by binding progesterone using anti-progesterone (anti-P4) coated polysterene tube. The next stage was examining the bioactivity of IGF-I and estrogen produced in this study as supplement media at fertilization and culture media by observation of embryo cleavage (2-8 cells embryo) and embryo develop into morula stage (6 days after in vitro fertilization). The last stage was examining is bioactivity test of IGF-I and estrogen mitogenic agent with observation reduction the occurence apoptosis of embryo in in vitro fertilization result.
Statistical analysis using factorial F-test (Anava) showed that in Stage 1 the mean of cumulus cell culture cell number was 62.66% and was significantly higher (p<0.05) than those of the liver cells’, which was 58.44%. Double comparison test with 5% honesty significant difference (HSD) against culture periods showed that the highest concentration was 70.94% which was found in day-6 culture and the lowest concentration was 44.69% which was found in day-12 culture. There was a significant difference (p<0.05) between these two concentrations.
Results from Stage 2 showed that IGF-I concentration in liver cell culture media was 108.98 ng/ml and was higher to those of the cumulus cells’, which was 69.67 ng/ml.
Estrogen and progesterone concentrations in cumulus cell culture media were respectively 126.83 pg/ml and 3.21 ng/ml which were very significantly higher (p<0.01) than those of the liver cells’ (60.44 pg/ml and 0.89 ng/ml). The highest concentration of IGF-I and estrogen were respectively 175.53 ng/ml and 127.19 pg/ml which were found in day-6 culture while the lowest concentration of IGF-I and estrogen were respectively 18.94 ng/ml and 53.00 pg/ml which were found in day-12 culture. There were significant differences (p<0.05) between them. The highest progesterone concentration was 3.281 ng/ml which was found in day-3 culture while the lowest concentration was 1.100 ng/ml which was found in day-12 culture. These concentrations were significantly different (p<0.05). It was also found that IGF-I, estrogen and progesterone cells of origin and culture periods had a very significant interaction (p<0.01).
In Stage 3 statistical analysis using independent t-test showed that the effectiveness of the use of anti-P4 for the absorbtion of progesterone in liver and cumulus cell culture media were respectively 58.43% and 60.86% that were not significantly different (p>0.05).
The results of Stage 4 research showed that the mean number of embryos underwent cleavage into 2-8 cells in fertilization media contain IGF-1 and estrogen from liver and cumulus cells were respectively 40.87% and 40.23% which were not differ significantly (p>0.05). Meanwhile the mean number of embryos underwent cleavage before and after absorbtion using antiprogesterone were 27.48% and 53.61% respectively which were significantly different (p<0.01). The mean number of embryos develop into morula in the culture contained IGF-1 growth factor and estrogen from liver and cumulus cells were 17.14% and 16.18% which were not different respectively significantly (p>0.05). Meanwhile, embryos developed into morula as supplemen media before and after absorbtion were respectively 5.73% and 27.59% which were significantly different (p<0.01).
Results from Stage 5 research were the mean of apoptosis incidence in embryos cultured in media contain growth factor IGF-1 and estrogen from monolayer culture of liver (P1) and cumulus cells (P2) respectively 23.72% and 31.01% which were not significantly different (p>0.05). Meanwhile embryos cultured in media with no IGF-1
and estrogen (P0) was 70.58% which were signicantly different to (P1) and (P2) (p<0.05).
From the result of the research it could be conducted that IGF-1 and estrogen which were mitogenic materials and progesterone which was an antimitogenic material could be produced through monolayer culture of liver and cumulus cells of cattle, and progesterone could be separated from IGF-1 and estrogen by binding/absorbtion using antiprogesterone (antiP4). Growth factor IGF-1 and estrogen from in vitro monolayer culture of liver and cumulus cells could be used as a supplementation media for bovine in vitro fertilization and embryo culture. Therefore resulted better cleavage and embryo development and inhibit the occurence of apoptosis in in vitro derived bovine embryos.
RINGKASAN
IDENTIFIKASI DAN UJI BIOAKTIVITAS GROWTH FACTOR DAN HORMON STEROID SEKS HASIL BIAKAN MONOLAYER SEL HEPAR DAN SEL
KUMULUS SAPI
Penerapan bioteknologi merupakan upaya meningkatkan efisiensi reproduksi ternak terutama guna mendapatkan ternak dengan kualitas dan kuantitas yang baik. Embrio transfer merupakan salah satu cara yang dipandang efisien dan efektif dalam bidang reproduksi. Keberhasilan hal tersebut membutuhkan embrio yang berkualitas. Produksi embrio secara in vivo terbatas oleh kemampuan ternak betina donor untuk menghasilkan embrio. Untuk itu produksi embrio secara in vitro merupakan suatu alternatif produksi embrio.
Faktor pertumbuhan atau Growth Factor yaitu Insulin-like Growth Factor-1 (IGF-1) dan hormon estrogen merupakan bahan yang bersifat mitogenik telah terbukti dapat digunakan untuk merangsang dan mengatur pertumbuhan sel.
Insulin-like Growth Factor-1 (IGF-1) atau somatomedin adalah polipeptida growth factor yang disekresikan oleh hati dan berbagai jaringan sebagai respon dari rangsangan growth hormon. IGF-1 tersusun atas 70 asam amino dengan berat molekul 7649 Dalton atau 7,65 kDa. Hormon estrogen adalah termasuk dalam hormon steroid, terutama diproduksi oleh folikel yang sedang berkembang di dalam ovarium, korpus luteum, dan plasenta dengan berat molekul 300 – 400 Dalton (272,3 Dalton). IGF-1 dan estrogen
bekerja secara parakrin, autokrin dan endokrin dalam merangsang aktivitas mitogenik pada sel yang sedang mengalami proliferasi.
Hepar dan ovarium merupakan bahan yang dapat memproduksi IGF-1 dan estrogen. Hepar merupakan penghasil utama IGF-1, sedangkan ovarium merupakan penghasil utama steroid seks estrogen. Tetapi keduanya dapat dihasilkan oleh organ tersebut dan dapat dikembangkan melalui kultur monolayer dengan penambahan bahan prekursor berupa Foetal Calf Serum (FCS) atau Bovine Serum Albumin (BSA) pada biakan monolayer yang digunakan.
Berdasarkan uraian tersebut, maka telah dilakukan penelitian dengan tujuan untuk memproduksi faktor pertumbuhan IGF-1 dan steroid seks estrogen dan progesteron dari cairan hasil kultur monolayer sel hepar dan sel kumulus dengan memanfaatkan bahan berupa hepar dan ovarium sapi yang dipotong di Rumah Potong Hewan (RPH).
Penelitian ini dilakukan secara bertahap, tahap 1 adalah pembuatan dan pemanenan cairan hasil kultur monolayer sel hepar dan sel kumulus. Kultur sel hepar dibuat dengan menggerus hepar kemudian dilakukan tripsinasi, kemudian disentrifugasi berulang-ulang sehingga diperoleh sel hepar (hepatosit). Sel kumulus diperoleh dengan cara aspirasi folikel dengan diameter 2-6 mm. Kemudian sel hepar dan sel kumulus masing-masing dikultur dalam media TCM 199 + FCS 10 % + BSA 10 %. Konsentrasi sel kultur adalah 1,9x106 sel/ml media, lalu diinkubasi dalam inkubator 38,5oC 5 % CO2 selama 3, 6, 9, dan 12 hari. Tahap ke 2 adalah pengukuran konsentrasi IGF-1 dalam cairan hasil kultur dengan teknik Immuno Radio Metric Assay (IRMA), sedangkan konsentrasi estrogen dan progesteron diukur dengan teknik Radio Immuno Assay (RIA). Tahap ke 3 adalah pemisahan/absorbsi progesteron yang merupakan bahan antimitogenik dari IGF-1 dan estrogen yang merupakan bahan mitogenik dengan cara mengikat progesteron dengan antiprogesteron (antiP4) dalam tabung coated polyesterene. Tahap selanjutnya adalah menguji bioaktivitas faktor pertumbuhan IGF-1 dan estrogen produk penelitian sebagai suplemen media pada fertilisasi dan kultur embrio in vitro dengan melihat adanya cleavage dan perkembangan embrio sampai stadium morula (6 hari setelah fertilisasi in vitro). Tahap terakhir adalah menguji bioaktivitas IGF-1 dan estrogen sebagai bahan yang bersifat mitogenik dan antiapoptotik dengan melihat penurunan kejadian apoptosis pada embrio hasil fertilisasi in vitro.
Berdasarkan analisis statistik dengan uji F (Anava) Faktorial, hasil penelitian tahap 1 menunjukkan bahwa rerata jumlah sel hasil kultur sel kumulus adalah 62,66% dan lebih tinggi daripada sel hepar yaitu 58,44% dengan signifikansi (p<0,05). Berdasarkan uji perbandingan honesty significant different (HSD) 5% terhadap lama kultur, kadar tertinggi diperoleh dari lama kultur 6 hari yaitu 70,94% dan terendah pada lama kultur kultur 12 hari yaitu 44,69% dan berbeda signifikan (p<0,05).
Hasil penelitian tahap 2 menunjukkan bahwa, konsentrasi IGF-1 hasil kultur monolayer sel hepar adalah 108,98 ng/ml dan lebih tinggi dari pada sel kumulus yaitu 69,67 ng/ml, sedangkan konsentrasi estrogen dan progesteron hasil kultur sel kumulus masing-masing adalah 126,83% pg/ml dan 3,21 ng/ml lebih tinggi daripada sel hepar yaitu 60,44 pg/ml dan 0,89 ng/ml, dan menunjukkan perbedaan sangat signifikan (p<0,01). Konsentrasi IGF-1 dan estrogen berdasarkan perbedaan lama inkubasi diperoleh hasil tertinggi dari lama inkubasi 6 hari masing-masing yaitu 175,53 ng/ml dan 127,19 pg/ml, sedangkan konsentrasi terendah pada inkubasi 12 hari masing-masing 18,94 ng/ml dan 53,00 pg/ml, dan berbeda secara signifikan (p<0,05). Konsentrasi progesteron berdasarkan lama masa inkubasi diperoleh hasil tertinggi dari kultur 3 hari yaitu 3,28 ng/ml, sedangkan terendah dari kultur 12 hari yaitu 1,10 ng/ml dan berbeda secara signifikan (p<0,05). Diketahui pula bahwa konsentrasi IGF-1, estrogen dan progesteron antara asal sel dan lama kultur menunjukkan interaksi yang sangat signifikan (p<0,01).
Hasil penelitian tahap 3 berdasarkan analisis statistik dengan uji t tak berpasangan (independent t test) menunjukkan bahwa efektivitas absorbsi progesteron dengan antiprogesteron antara sel hepar dan sel kumulus adalah 58,43 % dan 60,86% dan tidak berbeda secara signifikan (p>0,05).
Hasil penelitian tahap 4 menunjukkan bahwa rerata jumlah embrio yang mengalami cleavage 2 – 8 sel pada media fertilisasi mengandung IGF-1 dan estrogen antara sel hepar dan sel kumulus, masing-masing adalah sebesar 40,87 % dan 40,23 % yang tidak berbeda secara signifikan (p>0,05). Sedangkan antara sebelum dan sesudah absorbsi dengan antiprogesteron adalah 27,48 % dan 53,61 %, yang berbeda sangat signifikan (p<0,01). Rerata jumlah embrio yang berkembang menjadi stadium morula pada kultur yang mengandung faktor pertumbuhan IGF-1 dan estrogen antara sel hepar dan sel kumulus,
masing-masing adalah 17,14 % dan 16,18 % yang tidak berbeda secara signifikan (p>0,05). Sedangkan embrio yang berkembang menjadi morula pada media sebelum dan sesudah absorbsi dengan antiprogesteron, masing-masing adalah 5,73 % dan 27,59 %, yang berbeda sangat signifikan (p<0,01).
Hasil penelitian tahap 5 adalah rerata kejadian apoptosis pada embrio yang dikultur pada media yang mengandung growth factor IGF-1 dan estrogen hasil kultur monolayer sel hepar (P1) dan sel kumulus (P2), masing-masing adalah 23,72 % dan 31,01 %, yang tidak berbeda secara signifikan (p>0,05). Sedangkan embrio yang dikultur pada media yang tidak mengandung IGF-1 dan estrogen (P0) adalah sebesar 70,56 % dan berbeda secara signifikan dengan (P1) maupun (P2) (p<0,05).
Dari hasil penelitian dapat disimpulkan bahwa IGF-1 dan estrogen yang merupakan bahan bersifat mitogenik, serta progesteron yang bersifat antimitogenik dapat diproduksi melalui kultur monolayer sel hepar dan sel kumulus sapi, dan progesteron dapat dipisahkan dari IGF-1 dan estrogen dengan cara mengikat/mengabsorbsi dengan antiprogesteron (antiP4). Faktor pertumbuhan IGF-1 dan estrogen hasil kultur monolayer sel hepar dan sel kumulus secara in vitro dapat digunakan sebagai suplemen media pada fertilisasi dan kultur embrio sapi in vitro, sehingga menghasilkan cleavage dan perkembangan embrio yang lebih baik, dan dapat menghambat kejadian apoptosis pada embrio sapi hasil fertilisasi in vitro.
