Characteristics of rat round spermatids differentiated from spermatogonial cells during co-culture with Sertoli cells, assessed by flow cytometry, microinsemination and RT-PCR.
The present study was undertaken to investigate whether rat spermatogonial stem cells can differentiate into developmentally competent round spermatids during co-culture with Sertoli cells. Type-A spermatogonia and Sertoli cells were prepared from 7-d-old Wistar-strain male rats, and seeded at 4 x 10(6) cells/ 4 mL/35-mm dish (Day 0). They were co-cultured at 37 degrees C for 3 d and at 34 degrees C for the subsequent 7d in 5% CO(2)/air. Round spermatid-like cells (approximately 15 microm in diameter) were first observed on Day 5. A flow cytometric analysis showed that a single peak of haploid cells was detected in the cell populations harvested on Day 10. The participation of the spermatid-like cells to full-term development was examined by microinjection into activated oocytes. The oviductal transfer of 143 microinseminated oocytes resulted in only 8 implantation sites (6%), but no viable offspring. The expression of the round spermatid-specific marker gene, PRM-2, was confirmed in the Day 10 cell population by RT-PCR; however, no mRNA of two other haploid makers, TP1 or TP2, was detected. These results suggested that rat type-A spermatogonial cells underwent meiosis during the primary co-culture with the Sertoli cells, based on morphology, flow cytometry and PRM-2 expression, but the normality of the spermatid-like cells was not supported by microinsemination and TP1/2 expression.
Iwanami
Yoshihito
Y
Graduate School of Science and Technology, Shinshu University, Ueda, Nagano 386-8567, Japan.
Kobayashi
Toshihiro
T
Kato
Megumi
M
Hirabayashi
Masumi
M
Hochi
Shinichi
S
D000818:Animals; D000831:Animals, Newborn; D002454:Cell Differentiation; D002478:Cells, Cultured; D002868:Chromosomal Proteins, Non-Histone / Q000032:analysis / Q000096:biosynthesis; D018920:Coculture Techniques; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary*; D005434:Flow Cytometry / Q000379:methods; D008297:Male; D009687:Nuclear Proteins / Q000032:analysis / Q000096:biosynthesis; D009865:Oocytes / Q000254:growth & development; D011003:Ploidies; D011479:Protamines / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary; D012708:Sertoli Cells / Q000166:cytology / Q000502:physiology*; D020554:Sperm Injections, Intracytoplasmic / Q000379:methods; D013087:Spermatids / Q000145:classification / Q000166:cytology / Q000254:growth & development*; D013093:Spermatogonia / Q000166:cytology / Q000254:growth & development*
D002868:Chromosomal Proteins, Non-Histone; D017931:DNA Primers; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011479:Protamines; C489504:Tnp2 protein, rat; C068621:protamine 2; C053886:spermatid transition proteins