PMID- 11514353
OWN - NLM
STAT- MEDLINE
DCOM- 20011204
LR  - 20220318
IS  - 0006-3363 (Print)
IS  - 0006-3363 (Linking)
VI  - 65
IP  - 3
DP  - 2001 Sep
TI  - In vitro production of haploid germ cells from fresh or frozen-thawed testicular 
      cells of neonatal bulls.
PG  - 873-8
AB  - Improved methods for culturing spermatogenic cells will facilitate the study of 
      spermatogenesis, treatment of male factor infertility, and genetic modification 
      of the male germ line. The objective of this study was to develop a procedure for 
      achieving male germ cell progression through meiosis in vitro. Testes from 
      3-day-old bulls were decapsulated and seminiferous tubules were dissociated 
      enzymatically to recover Sertoli and germ cells. Dissociated cells were 
      reaggregated by phytohemagglutinin and encapsulated by calcium alginate, then 
      cultured for up to 14 wk in modified Dulbecco modified Eagle medium/F12 (32 
      degrees C, 5% CO(2) in air). At 2, 5, and 10 wk, cultured cells were examined and 
      evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and 
      Northern blot analysis for protamine-2 (PRM-2) and transition protein-1 (TP-1) 
      mRNA, expressed specifically in round spermatids. Ploidy was characterized by 
      flow cytometric analysis of DNA content of cultured cells. Only Sertoli cells and 
      gonocytes were observed in seminiferous tubules of 3-day-old testes. By 10 wk of 
      culture, small spherical cells (7-10 microm) were apparent at the margin of cell 
      associations in culture. Following RT-PCR and Northern blot analysis, specific 
      bands corresponding to PRM-2 and TP-1 were detected only in adult testis RNA or 
      after 10 wk of culture. Based on flow cytometry, a haploid population of cells 
      appeared in vitro that was not in 3-day-old bull testis. The novel culture system 
      developed in this study is the first to promote differentiation of gonocytes to 
      presumptive spermatids in vitro based on the expression of spermatid-specific 
      genes.
FAU - Lee, D R
AU  - Lee DR
AD  - Department of Animal Science, Cornell University, Ithaca, New York 14853, USA.
FAU - Kaproth, M T
AU  - Kaproth MT
FAU - Parks, J E
AU  - Parks JE
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Biol Reprod
JT  - Biology of reproduction
JID - 0207224
RN  - 0 (Chromosomal Proteins, Non-Histone)
RN  - 0 (Protamines)
RN  - 0 (RNA, Messenger)
RN  - 0 (protamine 2)
RN  - 0 (spermatid transition proteins)
RN  - 9007-49-2 (DNA)
SB  - IM
MH  - Animals
MH  - *Animals, Newborn
MH  - Blotting, Northern
MH  - Cattle
MH  - Cells, Cultured
MH  - Chromosomal Proteins, Non-Histone/genetics
MH  - Cryopreservation
MH  - DNA/analysis
MH  - Flow Cytometry
MH  - *Haploidy
MH  - Hot Temperature
MH  - Male
MH  - Meiosis
MH  - Protamines/genetics
MH  - RNA, Messenger/analysis
MH  - Reverse Transcriptase Polymerase Chain Reaction
MH  - Seminiferous Tubules/cytology
MH  - Spermatozoa/*cytology
MH  - Testis/*cytology
EDAT- 2001/08/22 10:00
MHDA- 2002/01/05 10:01
CRDT- 2001/08/22 10:00
PHST- 2001/08/22 10:00 [pubmed]
PHST- 2002/01/05 10:01 [medline]
PHST- 2001/08/22 10:00 [entrez]
AID - 10.1095/biolreprod65.3.873 [doi]
PST - ppublish
SO  - Biol Reprod. 2001 Sep;65(3):873-8. doi: 10.1095/biolreprod65.3.873.