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Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos
Published on: February 22, 2016
Gonadal expression of c-kit encoded at the W locus of the mouse
1Department of Cell Biology and Anatomy, Cornell University Medical College, New York, NY 10021.
Abstract:
Recently, it has been shown that the c-kit proto-oncogene is encoded at the white spotting (W) locus in mice. Mutations of this gene cause depletion of germ cells, some hematopoietic cells and melanocytes. In order to define further the role of c-kit in gametogenesis, we have examined its expression in late fetal and postnatal ovaries and in postnatal testis. By RNA blot analysis, c-kit transcripts were not detected in late fetal ovaries but appeared at birth. The relative amount reached a maximum in ovaries of juvenile mice, and decreased in adult ovaries. c-kit transcripts were present in increasing amounts in isolated primordial, growing and full-grown oocytes, as well as in ovulated eggs. Little was detected in early 2-cell embryos and none in blastocysts. In situ hybridization revealed c-kit transcripts in a few oocytes of late fetal ovaries and in all oocytes (from primordial to full-grown) in ovaries from juvenile and adult mice. Expression was also observed in ovarian interstitial tissue from 14 days of age onward. Using indirect immunofluorescence, the c-kit protein was detected on the surface of primordial, growing and full-grown oocytes, as well as on embryos at the 1- and 2-cell stages; little remained in blastocysts. In situ hybridization analysis of testes from mice of different ages demonstrated expression in spermatogonia from 6 days of age onward. Using information provided by determining the stage of the cycle of the seminiferous epithelium for a given tubule and by following the age dependence of labeling, it was concluded that the period of expression of c-kit extends from at least as early as type A2 spermatogonia through type B spermatogonia and into preleptotene spermatocytes. Leydig cells were labelled at all ages examined. The expression pattern in oocytes correlates most strongly with oocyte growth and in male germ cells with gonial proliferation.
Insights
The c-kit proto-oncogene is crucial for germ cell development in mice. Its expression in ovaries and testes correlates with oocyte growth and spermatogonial proliferation, respectively.
Area of Science:
- Reproductive Biology
- Developmental Biology
- Molecular Genetics
Background:
- The c-kit proto-oncogene, located at the white spotting (W) locus in mice, is implicated in germ cell development.
- Mutations in c-kit lead to deficiencies in germ cells, hematopoietic cells, and melanocytes.
Purpose of the Study:
- To investigate the expression patterns of c-kit in the ovaries and testes of mice during different developmental stages.
- To further elucidate the role of c-kit in gametogenesis.
Main Methods:
- RNA blot analysis to detect c-kit transcripts.
- In situ hybridization to localize c-kit expression in ovarian and testicular tissues.
- Indirect immunofluorescence to detect c-kit protein on cell surfaces.
Main Results:
- c-kit transcripts and protein are present in oocytes from primordial to full-grown stages, correlating with oocyte growth.
- Expression is observed in spermatogonia (Type A2 through Type B) and preleptotene spermatocytes in testes, linked to gonial proliferation.
- c-kit is also found in ovarian interstitial tissue and Leydig cells.
Conclusions:
- c-kit plays a significant role in both female and male gametogenesis in mice.
- Its expression pattern suggests involvement in critical stages of germ cell proliferation and maturation.
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