Isolation and identification of germ cells from fetal bovine ovaries

Abstract

Gonadal cell suspensions were made from bovine fetuses of 35-55-, 56-80-, and 80-130-day age groups corresponding to the periods predominated by primordial germ cells (PGCs), oogonia, and meiotic cells, respectively. Germ cells identified on morphological criteria prior to their isolation from suspensions were compared histochemically and morphologically with cells in cryosections, impression smears, and semithin sections of similar gonads. Oocytes were distinguished by their chromosomal configurations in cell spreads. In suspensions from 35-55-day fetuses, cells considered to be PGCs stood out by their size, large nucleus, intracytoplasmic vesicles, and occasional blebbing. The somatic cells were smaller and contained little cytoplasm and few vesicles. In bovine gonads, in contrast to murine gonads, alkaline phosphatase (AP) activity was not specific enough to identify germ cells once they had entered the gonad. In ovaries from the 56-80-day age group, cells similar to PGCs, but slightly larger and with more cytoplasmic vesicles, were identified as oogonia. The cytoplasmic vesicles stained positively for lipid. In ovaries of 80-130-day fetuses, oogonia, oocytes, degenerating germ cells, and multinucleate germ cells were recognized. Degenerating germ cells exhibited a variety of morphological characteristics and were consistently positive for acid-phosphatase activity. Binucleate germ cells appeared around day 85 of gestation, while multinucleate germ cells were seen from day 95. It was concluded that bovine mitotic germ cells can be isolated from gonadal cell suspensions and that the best time to recover them is between 50 and 70 days of gestation.

Cloning in cattle: from embryo splitting to somatic nuclear transfer

“…Such cells are known to be pluripotent at least in the mouse. Gonial cells are diploid and can be isolated in large numbers from bovine foetal gonads [28]. The pluripotency of bovine gonial cells has been tested through nuclear transfer by three separate laboratories, leading to the temporary conclusion that germ cells can direct in vitro development up to blastocysts in some cases (2-4 %), but no live offspring could be obtained after transfer to recipient heifers.…”

Effects of Protease Inhibitors and Antioxidants on In Vitro Survival of Porcine Primordial Germ Cells1

“…Pig [17], cattle [7,18], rabbit [7,19], and rat [7,20] PGCs have been collected and characterized by their morphology and immunohistochemistry. We and others have previously shown that porcine EG cell lines can be isolated [21][22][23], genetically transformed [21], and contribute to chimera development in the pig [21][22][23].…”

Segregation of Retinoic Acid Effects on Fetal Ovarian Germ Cell Mitosis Versus Apoptosis by Requirement for New Macromolecular Synthesis*

“…In H/E-stained sections, cells possessing lightly stained round nuclei along with a maintenance of cytoplasmic volume and easily discernible spherical plasma membranes were considered nonapoptotic, whereas cells showing nuclear condensation (basophilia), cytoplasmic shrinkage, and convoluted plasma membranes were considered apoptotic (21). As previously established for studies of the fetal ovary (22)(23)(24), germ cells were distinguished from somatic cells based on differences in cellular size (germ cells being much larger than somatic cells) and morphology (germ cells and their nuclei being spherical as opposed to somatic cells being squamous with flattened nuclei). Furthermore, morphological identification of germ cells was confirmed by alkaline phosphatase staining in preliminary unpublished studies conducted during the validation of this culture model (17).…”