Wahono Esthi Prasetyaningtyas
DepartemenAnatomi, Fisiologi dan Farmakologi, Fakultas Kedokteran Hewan, Institut Pertanian Bogor, Bogor

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PF-17 THE DEVELOPMENT OF CRUDE TESTICULAR CELLS IN IN VITRO CULTURE Prasetyaningtyas, Wahono Esthi; Karja, Ni Wayan Kurniani; Agungpriyono, Srihadi; Fahrudin, Mokhamad
Hemera Zoa Proceedings of the 20th FAVA & the 15th KIVNAS PDHI 2018
Publisher : Hemera Zoa

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Abstract

Spermatogenesis is a continuous process in which spermatogonial stem cells (SSC) develop into specific germ cells before terminally differentiating to form spermatozoa.  The process is supported by Sertoli cells, which are in close contact with germ cells in the seminiferous tubules. Sertoli cells provide essential hormonal signals, nutrients, and physical support to germ cells for successful spermatogenesis.The crude testicular cells (CTC) contains many cell types, like Sertoli cell, Leydig cell, spermatogonial stem cell (SSC), spermatocyte and other testicular somatic cells (Shah et all. 2016). Testicular cells are believed to secrete various growth factors that induced the spermatogenesis process.  The spermatogonial stem cells are unique population of cells in the male testis, which dual function.  First self-renewing their population to maintain the number of stem cells, secondary function is differentiating into spermatids in testis (Wang et al.  2015).Spermatogenic cells differentiation  needed the similar microenvironment in vivo spermatogenesis.  The essential nutrients was collected from healty culture and the culture contained mixed population of cells both the somatic cells and spermatogenic cells.  To identification the spermatogenic cells using Periodic Acid Schifft (PAS) staining (Chang et al. 2011). The present study examined the development of crude testicular cells using PAS staining.
EVALUATION OF RAT LEYDIG CELL CULTURE COLLECTED WITH NYCODENZ GRADIENT IN PRODUCING TESTOSTERONE IN VITRO Kaiin, Ekayanti Mulyawati; Prasetyaningtyas, Wahono Esthi
Jurnal Kedokteran Hewan Vol 11, No 4 (2017): J. Ked. Hewan
Publisher : Universitas Syiah Kuala

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (285.769 KB) | DOI: 10.21157/j.ked.hewan.v11i4.3984

Abstract

 The aim of this study was to evaluate the ability of rat?s Leydig cells collected with Nycodenz gradient in producing testosterone in vitro. Leydig cells were collected using 5 column of Nycodenz gradient (4, 8, 10, 12, and 15%) and cells were evaluated regarding its concentration, viability, and purity of Leydig cells. Media used to cultured Leydig cells were Dulbecco?s Modified Eagle?s Medium (DMEM)+10% newborn calf serum (NBCS); DMEM+10% NBCS+2,5 IU/mL human chorionic gonadotrophin (hCG); DMEM+10% NBCS+ 5 µg/mL insulin, 10 µg/mL transferrin, and 5 µg/mL Se (ITS); DMEM+10% NBCS+hCG+ITS at 5% CO2 incubator with temperature of 37.5° C for 3 days. Culture medium was collected every day for testosterone analysis with enzyme-linked immunosorbent assay (ELISA). By adding ITS to the medium, Leydig cells concentration was significantly increased (8.92x106  cells/mL) compared to medium with serum (7.74x106 cells/mL) or hCG (7.68x106 cells/mL) (P 0.05). ITS and hCG in medium significantly increased Leydig cells concentration (10.40x106 cells/mL) at day 3 of culture (P 0.05). The result of parallelism test showed that the assay obtained good validity to measure testosterone concentration in culture medium. Testosterone in medium was detected at 1.80-2.60 ng/mL at day 1 of culture. In conclusion, Leydig cells collected with Nycodenz gradient had no effect to testosterone secretion from Leydig cells in vitro.
TINGKAT PROLIFERASI PRIMORDIAL GERM CELLS SECARA IN VITRO DALAM MEDIUM KULTUR DENGAN PENAMBAHAN LEUKEMIA INHIBITORY FACTOR (IN VITRO PROLIFERATION RATE OF MICE PRIMORDIAL GERM CELLS IN THE CULTURE MEDIUM WITH ADDITION OF LEUKEMIA INHIBITORY FACTOR) Prasetyaningtyas, Wahono Esthi; Karja, Ni Wayan Kurniani; Agungpriyono, Srihadi; Fahrudin, Mokhamad
Jurnal Veteriner Vol 20 No 4 (2019)
Publisher : Faculty of Veterinary Medicine, Udayana University and Published in collaboration with the Indonesia Veterinarian Association

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (136.304 KB) | DOI: 10.19087/jveteriner.2019.20.4.526

Abstract

Primordial germ cells (PGCs) are precursors for gamete cells. The totipotency of PGCs allows them to be used as a model for studying cancer and infertility. The study aimed to examine the characteristics of the mice fetus as a source of PGCs, proliferation rate of PGC and the role of LIF in vitro culture of PGCs. This study used genital ridges from 26 fetuses at 13.5 days post-coital (dpc) to isolate the PGCs. Genital ridges dissociation using 0.1% of trypsin and in vitro culture was carried out using the Dulbecco Modified Eagle Medium (DMEM) and incubated at 37 0C and 5% CO2 atmosphere. The fetus was measured and weighed to determine the normal development of the fetus and continued with the identification of the genital ridges after laparotomy performed under a stereomicroscope. Proliferation rate was measured by calculating Population Doubling Time (PDT), and cell viability was observed after in vitro culture for six days.  The effect of  adding 1000 IU/ml of leukemia inhibitory factor (LIF) was evaluated from two types of treatment in the medium, 1) DMEM added with 15% of fetal calf serum (FCS) (DMEM + S15%) and 2), DMEM was supplemented with 15% of FCS and 1000 IU/ml LIF (DMEM + S15% + LIF1000 IU/ml). Immunohistochemistry staining was carried out on the third-, sixth- and ninth-day of culture to detect the expression of Oct-4 in the PGCs, then cells were counted. The results showed that the fetus as a source of PGCs had normal development. The fetal sizes were 11 mm, and male and female genital ridges could be distinguished by morphology at the age of 13.5 dpc. The proliferation of PGCs was relatively slow with a 1.3 day PDT value with the viability of around 85%.  Culture of PGCs with DMEM + S15% treatment showed the percentage of PGCs that expressing Oct-4 decreased from the third day of culture to the ninth day of culture. The culture of PGCs in DMEM + S15% + LIF 1000 IU / ml treatment showed that the percentage of PGCs that expressed Oct 4 increased on the sixth day of culture and decreased on the ninth day of culture. It can be concluded that the addition of LIF can maintain the number of PGCs until the sixth day of culture. LIF is thought to play a role in the regulation of proliferation of PGCs through receptors of LIF (RLIF) and glicoprotein (gp) 130 receptors. 
Status of ram spermatozoa DNA after freeze-drying process Saili, Takdir; Prasetyaningtyas, wahono Esthi; Setiadi, Mohamad Agus; AgungPriyono, Srihadi; Boediono, Arief
Indonesian Journal of Animal and Veterinary Sciences Vol 11, No 3 (2006)
Publisher : Indonesian Animal Sciences Society

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (260.586 KB) | DOI: 10.14334/jitv.v11i3.528

Abstract

The process of freeze drying caused detrimental effect on plasma membrane and acrosome of the spermatozoa, even it potentially could alter the chromatin and DNA integrities. On the other hand, DNA integrity is essential for spermatozoa to participate in pronucleus formation during fertilization event. Therefore the evaluation of DNA integrity should be carried out to study the effect of freeze drying process. EDTA, EGTA, and PBS were used as dilution media of spermatozoa prior to freeze drying process to protect the DNA. Toluidine blue staining and comet assay methods were used to evaluate the alteration on chromatin and DNA integrities of spermatozoa, respectively. The results revealed that the highest compacted chromatin after 6 months storage of freeze-dried spermatozoa were observed from EGTA-3 (98%) and EGTA-1 (97%) treatments that had significant differences compared to all PBS treatments (90-92%), but not for fresh spermatozoa (100%). Whereas, the highest compacted DNA integrity of freeze-dried spermatozoa were observed from EGTA-2 (92%) and EGTA-3 (92%) but had no significant differences compared to other treatments including fresh spermatozoa (97%). These results demonstrate that EDTA and EGTA tend to be able to protect chromatin and DNA integrities of ram spermatozoa during freeze-drying and storage compared to PBS. Key Words: Freeze-Drying, Spermatozoa, DNA, Toluidine Blue, Comet Assay
Status of ram spermatozoa DNA after freeze-drying process Saili, Takdir; Prasetyaningtyas, wahono Esthi; Setiadi, Mohamad Agus; AgungPriyono, Srihadi; Boediono, Arief
Jurnal Ilmu Ternak dan Veteriner Vol 11, No 3 (2006): SEPTEMBER 2006
Publisher : Indonesian Animal Sciences Society

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (260.586 KB) | DOI: 10.14334/jitv.v11i3.528

Abstract

The process of freeze drying caused detrimental effect on plasma membrane and acrosome of the spermatozoa, even it potentially could alter the chromatin and DNA integrities. On the other hand, DNA integrity is essential for spermatozoa to participate in pronucleus formation during fertilization event. Therefore the evaluation of DNA integrity should be carried out to study the effect of freeze drying process. EDTA, EGTA, and PBS were used as dilution media of spermatozoa prior to freeze drying process to protect the DNA. Toluidine blue staining and comet assay methods were used to evaluate the alteration on chromatin and DNA integrities of spermatozoa, respectively. The results revealed that the highest compacted chromatin after 6 months storage of freeze-dried spermatozoa were observed from EGTA-3 (98%) and EGTA-1 (97%) treatments that had significant differences compared to all PBS treatments (90-92%), but not for fresh spermatozoa (100%). Whereas, the highest compacted DNA integrity of freeze-dried spermatozoa were observed from EGTA-2 (92%) and EGTA-3 (92%) but had no significant differences compared to other treatments including fresh spermatozoa (97%). These results demonstrate that EDTA and EGTA tend to be able to protect chromatin and DNA integrities of ram spermatozoa during freeze-drying and storage compared to PBS. Key Words: Freeze-Drying, Spermatozoa, DNA, Toluidine Blue, Comet Assay
BLACK SEED (NIGELLA SATIVA) EXTRACT INDUCE IN VITRO PROLIFERATION AND DIFFERENTIATION OF RAT PANCREATIC AND BONE CELLS (EKSTRAK JINTAN HITAM (NIGELA SATIVA) MENGINDUKSI PROLIFERASI DAN DIFERENSIASI SEL PANKREAS DAN SEL TULANG TIKUS SECARA IN VITRO) Prasetyaningtyas, Wahono Esthi; Romadhon, Deny Putra; Susana, Fitri; Djuwita, Ita; Mohamad, Kusdiantoro
Jurnal Veteriner Vol 17 No 3 (2016)
Publisher : Faculty of Veterinary Medicine, Udayana University and Published in collaboration with the Indonesia Veterinarian Association

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Abstract

Black seed (Nigella sativa), a medicinal plant, widely used for treating various diseases, includingdiabetes mellitus and osteoporosis. This study examined the proliferation and differentiation of pancreaticand bone cells of rat cultured in vitro in medium supplemented with N. sativa extracts (NS). Pancreaticand bone cells were isolated from five days old rat and cultured in Dulbecco modified eagle mediumsupplemented without NS (0%, as control), and with NS (0.05% and 0.5%, as treatment groups) in 5% CO2incubator at 37oC for seven days and observed for cell population doubling time (PDT); proportion anddiameter of Langerhans islets, osteoblast, and osteocyte; and proportion of Langerhans islets containingb cell expressing insulin secretion. The pancreatic b cells were observed using dithizone staining, while thebone cells using alizarin red staining. The result showed that supplementation of NS significantly (p<0.05)decreased the PDT of pancreatic and bone cells, increased the proportion and diameter of Langerhansislets, increased the proportion of expression the b cell producing insulin, and increased the diameter ofosteoblast. In conclusion, the supplementation of NS in culture medium improved the proliferation anddifferentiation of pancreatic and bone cells in vitro.
AH-25 SPERM MORPHOLOGY OF THE JAVAN MUNTJAK, MUNTIACUS MUNTJAK MUNTJAK Wahyuni, Sri; Gholib, Gholib; Prasetyaningtyas, Wahono Esthi; Adnyane, I Ketut Mudite; Agungpriyono, Srihadi; Hamny, Hamny; Jalaluddin, Muhammad; Sabri, Mustafa; Akmal, Muslim; Agil, Muhammad; Yusuf, Tuty Laswardi
Media Veteriner Proceedings of The 5th Congress of Asian Association of Veterinary Anatomists (Asian AVA) 2015
Publisher : Media Veteriner

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Abstract

Sperm Morphology of the Javan Muntjak,  Muntiacus muntjak muntjak
Biological Analysis of Leydig Cells-Conditioned Medium To Support Rat Bone Marrow Mesenchymal Stem Cells Differentiation Kaiin, Ekayanti Mulyawati; Prasetyaningtyas, Wahono Esthi; Mohamad, Kusdiantoro; Djuwita, Ita; Yusuf, Tuty Laswardi; Setiadi, Mohamad Agus
ANNALES BOGORIENSES Vol 22, No 1 (2018): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14203/ab.v22i1.328

Abstract

The developed Leydig cells-conditioned medium (LCM) contains bioactive materials secreted by Leydig cells in vitro.  LCM was used to evaluate the ability of bone marrow mesenchymal stem cells differentiation. Bone marrow mesenchymal stem cells (1x 106 cell/ml) were cultured in : 1) DMEM supplemented with 10% NBCS as a control (M), 2) M supplemented with 10 ng/ml testosterone; 3) M supplemented with 50%  LCM ; 4) M supplemented with 50% LCM and 2.5 IU/ml hCG. Bone marrow mesenchymal stem cells that were cultured with  LCM has a positive reaction (57.4%) to histochemistry staining 3β-HSD and produced 1.87 ng/ml testosterone. Supplementation of hCG to LCM  increased the positive number of Leydig cells and testosterone production by 74.6% and 12.33 ng/ml (P<0.05). It can be concluded that Leydig cells-conditioned medium can support differentiation of bone marrow mesenchymal stem cells into Leydig cells.
ALLOTRANSPLANTASI TESTIS MENCIT MUDA SEBAGAI UPAYA PRESERVASI GONAD IN VIVO Prasetyaningtyas, Wahono Esthi; Mohamad, Kusdiantoro; Fahrudin, Mokhamad; Djuwita, Ita; Agungpriyono, Srihadi
Jurnal Ilmu Pertanian Indonesia Vol. 12 No. 1 (2007): Jurnal Ilmu Pertanian Indonesia
Publisher : Institut Pertanian Bogor

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Abstract

Cancer disease are not detected only at adult but also at young age. One therapy for cancer diseases is chemotherapy and radiation, that give a side effect of infertility in the gonad, therefore, it is necessary to preserve the gonad. Sperm collection from adult is easy but not from the young patients.Keyword: transpalantation, testis, mencit muda, spermatogenesis
BIOLOGICAL ANALYSIS OF LEYDIG CELLS-CONDITIONED MEDIUM TO SUPPORT RAT BONE MARROW MESENCHYMAL STEM CELLS DIFFERENTIATION Kaiin, Ekayanti Mulyawati; Prasetyaningtyas, Wahono Esthi; Mohamad, Kusdiantoro; Djuwita, Ita; Yusuf, Tuty Laswardi; Setiadi, Mohamad Agus
ANNALES BOGORIENSES Vol 22, No 1 (2018): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (457.602 KB) | DOI: 10.14203/ann.bogor.2018.v22.n1.20-26

Abstract

The developed Leydig cells-conditioned medium (LCM) contains bioactive materials secreted by Leydig cells in vitro.  LCM was used to evaluate the ability of bone marrow mesenchymal stem cells differentiation. Bone marrow mesenchymal stem cells (1x 106 cell/ml) were cultured in : 1) DMEM supplemented with 10% NBCS as a control (M), 2) M supplemented with 10 ng/ml testosterone; 3) M supplemented with 50%  LCM ; 4) M supplemented with 50% LCM and 2.5 IU/ml hCG. Bone marrow mesenchymal stem cells that were cultured with  LCM has a positive reaction (57.4%) to histochemistry staining 3?-HSD and produced 1.87 ng/ml testosterone. Supplementation of hCG to LCM  increased the positive number of Leydig cells and testosterone production by 74.6% and 12.33 ng/ml (P<0.05). It can be concluded that Leydig cells-conditioned medium can support differentiation of bone marrow mesenchymal stem cells into Leydig cells.