Cabrera M, Chan P J, Kalugdan T H, King A
Department of Gynecology and Obstetrics, Loma Linda University School of Medicine, California 92350, USA.
J Assist Reprod Genet. 1997 Feb;14(2):120-4. doi: 10.1007/BF02765781.
The purpose of this study was to determine whether exogenous DNA internalized into blastocysts after transference from DNA-carrier sperm are localized at the inner cell mass or trophoblast cells and to identify differences in uptake of exogenous DNA fragments by sperm due to unique DNA sequences.
Mouse blastocysts at the hatching stage were exposed to migrating human sperm cells carrying exogenous DNA fragments synthesized from the E6-E7 conserved gene regions of human papillomavirus (HPV) types 16 and 18. After an interaction period of 2 hr, the transfected blastocysts were washed several times to remove extraneous sperm and the blastocysts were dissected into groups of cells derived from the inner cell mass and trophoblasts. The cells were analyzed by polymerase chain reaction (PCR) for the presence of HPV DNA fragments. In the second part of the experiment, thawed donor (N = 10) sperm cells were pooled, washed, and divided into two fractions. The first (control) fraction was added with formalin and further divided and added with a 35S-radiolabeled G, A, T, or C sequencing mixture. The second fraction was similarly treated but the formalin step was omitted from the treatment. After an hour of incubation at 37 degrees C, the sperm specimens were washed several times by centrifugation and DNA extracted by the GeneReleaser method. The extracted DNA were processed on sequence gels, and the autoradiographs analyzed.
Mouse blastocysts transfected by carrier sperm with DNA from HPV types 16 and 18 showed localization of the HPV DNA to both the inner cell mass and trophoblast cells. Negative controls consisting of untreated human sperm and untreated mouse blastocysts did not reveal any evidence of HPV DNA. The positive sperm control generated expected DNA fragments from HPV types 16 and 18. In the second experiment, the intensities of the DNA fragments in the G, A, T, and C columns from low to high molecular weights were not different from the positive control bands. Band intensities of the four sequencing columns were similar. Formalin pretreatment of the sperm inhibited uptake of the DNA fragments from the smallest to the largest DNA molecules.
Exogenous DNA taken into blastocysts are localized to both the inner cell mass and trophoblast cells. Only live sperm exhibited the capacity to carry various sizes of exogenous DNA, suggesting the involvement of active cell membrane mechanism in the transference process. The results showed that DNA fragments terminating in any of the four nucleotides were equally taken up by the sperm cell. Fragments of DNA produced by the sequencing reaction failed to identify a unique DNA sequence that would facilitate or inhibit the sperm from taking up exogenous DNA.
本研究的目的是确定从携带DNA的精子转移后内化到囊胚中的外源DNA是定位于内细胞团还是滋养层细胞，并确定由于独特的DNA序列导致精子对外源DNA片段摄取的差异。
将孵化阶段的小鼠囊胚暴露于携带由人乳头瘤病毒（HPV）16型和18型的E6-E7保守基因区域合成的外源DNA片段的迁移人类精子细胞。经过2小时的相互作用期后，将转染的囊胚洗涤数次以去除多余的精子，并将囊胚解剖成源自内细胞团和滋养层的细胞群。通过聚合酶链反应（PCR）分析细胞中HPV DNA片段的存在情况。在实验的第二部分，将解冻的供体（N = 10）精子细胞汇集、洗涤并分成两部分。第一部分（对照）加入福尔马林，进一步分开并加入35S放射性标记的G、A、T或C测序混合物。第二部分进行类似处理，但处理过程中省略福尔马林步骤。在37℃孵育1小时后，通过离心将精子标本洗涤数次，并通过基因释放剂方法提取DNA。提取的DNA在序列凝胶上进行处理，并分析放射自显影片。
由携带来自HPV 16型和18型DNA的载体精子转染的小鼠囊胚显示HPV DNA定位于内细胞团和滋养层细胞。由未处理的人类精子和未处理的小鼠囊胚组成的阴性对照未显示任何HPV DNA的证据。阳性精子对照产生了来自HPV 16型和18型的预期DNA片段。在第二个实验中，从低分子量到高分子量的G、A、T和C列中的DNA片段强度与阳性对照带没有差异。四个测序列的带强度相似。精子的福尔马林预处理抑制了从最小到最大DNA分子的DNA片段摄取。
进入囊胚的外源DNA定位于内细胞团和滋养层细胞。只有活精子表现出携带各种大小外源DNA的能力，表明在转移过程中涉及活跃的细胞膜机制。结果表明，以四种核苷酸中的任何一种结尾的DNA片段被精子细胞同等摄取。测序反应产生的DNA片段未能鉴定出有助于或抑制精子摄取外源DNA的独特DNA序列。
