Female Primordial Germ Cells Research Articles

Differentiation of female chicken primordial germ cells into spermatozoa in male gonads.

In avian species, the developmental fate of different-sex germ cells in the gonads is unclear. The present study attempted to confirm whether genetically female germ cells can differentiate into spermatozoa in male gonads using male germline chimeric chickens produced by the transfer of primordial germ cells (PGC), and employing molecular biological methods. As a result of Southern hybridization, specific sequences of the W chromosome (the female specific sex chromosome in birds) were detected in the genomic DNA extracted from one out of four male germline chimeric chickens. When two-color in situ hybridization was conducted on the spermatozoa of this germline chimera, 0.33% (average) of the nuclei of each semen sample showed the fluorescent signal indicating the presence of the W chromosome. The present study shows that female PGC can differentiate into spermatozoa in male gonads in the chicken. However, the ratio of produced W chromosome-bearing (W-bearing) spermatozoa fell substantially below expectations. It is therefore concluded that most of the W-bearing PGC could not differentiate into spermatozoa because of restricted spermatogenesis.

Transfer of male or female primordial germ cells of quail into chick embryonic gonads.

Blood from an individual quail embryo at stages 13-16, when primordial germ cells (PGCs) were in circulation, was taken from its marginal vein and transfused into the marginal vein of a chick embryo at stages 13-16. Both donor and recipient embryos were cultured in vitro until day 8 of development and their sex was determined by morphological and histological observations of the gonads. Sections of recipient gonads were stained immunohistochemically with QCR1 monoclonal antibody positive for quail PGCs but negative for chick PGCs. Donor and recipient embryos were sexed in 17 pairs which included all four sex combinations. Transferred PGCs, either female-derived ZW type or male-derived ZZ type, were observed in the gonads of both sexes of 15 recipient embryos. The population of donor PGCs ranged from 20 to over 2500. In all four sex combinations, there was a higher population in the left than the right gonad of the embryos.

Sexual differentiation of chimeric chickens containing ZZ and ZW cells in the germline

The developmental fate of male and female cells in the ovary and testis was evaluated by injecting blastodermal cells from Stage X (Eyal-Gliadi and Kochav, 1976: Dev Biol 49:321-337) chicken embryos into recipients at the same stage of development to form same-sex and mixed-sex chimeras. The sex of the donor was determined by in situ hybridization of blastodermal cells to a probe derived from repetitive sequences in the W chromosome. The sex of the recipient was assigned after determination of the chromosomal composition of erythrocytes from chimeras at 10, 20, 40, and 100 days of age. If the sex chromosome complement of all of the erythrocytes was the same as that of blastodermal cells from the donor, the sex of the recipient was assumed to be the same as that of the donor. Conversely, if the sex-chromosome complement of a portion of the erythrocytes of the chimera differed from that of the donor blastodermal cells, the sex of the recipient was assumed to differ from that of the donor. Injection of male blastodermal cells into female recipients produced both male and female chimeras in equal proportions whereas injection of female cells into male recipients produced only by male chimeras. One phenotypically male chimera developed with a left ovotestis and a right testis although sexual differentiation was usually resolved into an unambiguous sexual phenotype during development when ZZ and ZW cells were present in a chimera. Donor cells contributed to the germline of 25-33% of same-sex chimeras whereas 67% of male chimeras produced by injecting male donor cells into female recipients incorporated donor cells into the germline. When ZW cells were incorporated into chimeric males, W-chromosome-specific, DNA sequences were occasionally present in DNA extracted from semen. To examine the potential of W-bearing spermatozoa to fertilize ova, males producing ZW-derived offspring and semen in which W-chromosome-specific DNA was detected by Southern analysis were mated to sex-linked albino hens. Since sex-linked albino female progeny were not obtained from this mating, it was concluded that the W-bearing sperm cells were unable to fertilize ova. The production of Z-derived, but not W-derived, offspring from ZW spermatogonia indicates that female primordial germ cells can become spermatogonia in the testes. In the testes, ZW spermatogonia enter meiosis I and produce functional ZZ spermatocytes. The ZZ spermatocytes complete the second meiotic division, continue to differentiate during spermiogenesis, and leave the seminiferous tubules as functional spermatozoa. By contrast, the WW spermatocytes do not appear to complete spermiogenesis and, therefore, spermatozoa bearing the W-chromosome are not produced. When cells from male embryos were incorporated into a female chimera, ZZ "oogonia" were included within the ovarian follicles and the chromosome complement of genetically male oogonia was processed normally during meiosis. Following ovulation, the male-derived ova were fertilized and produced normal offspring. This is the first reported evidence that genetically male avian germ cells can differentiate into functional ova and that genetically female germ cells can differentiate into functional sperm.

An immunohistochemical study of chick primordial germ cells using the mouse antiserum against chick embryonic gonads.

We established a mouse antiserum by immunizing BALB/c mice with sexually undifferentiated 6-day chick embryonic gonads. The antiserum had a higher affinity to the plasma membrane and cytoplasm of primordial germ cells (PGCs) than to the somatic cells in 6-day chick gonads. PGCs at this stage appear to have a higher specificity with more antigenic capacity than gonadal somatic cells; thus, the antiserum should be useful as a cell marker of chick PGCs. Subsequently, an immunohistochemical examination of PGCs settling in the genital ridges of chick embryos was carried out by the use of the antiserum. The reaction was clearly detected in the plasma membrane and cytoplasm of 5-day PGCs. After sexual differentiation, the reaction changed in distribution in females. At 8 days, it was limited to the perinuclear region of PGCs. In males, although the reaction persisted for longer period than in females, only a weak reaction was detectable at 9 days. These results suggest that the occurrence of periodic molecular changes may be different between male and female PGCs.

Evidence That the Mechanism of Prenatal Germ Cell Death in the Mouse Is Apoptosis

Using fluorescence-activated cell sorting combined with fluorescence microscopy the mechanism of embryonic germ cell death in the mouse has been shown to be apoptosis. Primordial germ cells (PGCs) from embryos at specific developmental stages have been analyzed, and cells with apoptotic morphology have been isolated by cell sorting. In the female, apoptotic oogonia at Day 13 and apoptotic oocytes at Days 15 and 17 were found. In the male, apoptotic cells were seen on Day 13 through Day 17. Apoptotic germ cells were not detected at Day 12 (combined male and female PGCs). Examination of sorted cells by fluorescence microscopy and by light microscopic analysis after alkaline phosphatase staining confirmed that the cells are apoptotic germ cells. Electron microscopy further confirmed that cells showing the morphological characteristics of apoptosis are present.

Changes in protooncogene expression correlated with general and sex-specific differentiation in murine primordial germ cells

Primordial germ cells (PGCs) of the mouse undergo key developmental transitions during embryonic days 12–15. On day 12 they complete migration into the gonads. They cease mitotic proliferation on day 13 and subsequently enter sex-specific pathways of development. The molecular mechanisms controlling these transitions are poorly understood, yet they are crucial to production of normal gametes later in life. We have used the polymerase chain reaction (PCR) to directly compare levels of expression of several protooncogenes proposed to be involved in control of cell proliferation and differentiation in proliferating and differentiating PGCs of both sexes over a 4 day time course. We report here that mRNA levels for nuclear protooncogenes c-myc, c-fos, and c-jun increase dramatically in both sexes from little or no detectable expression on day 12 to high expression on days 13–15. We observe c-kit message on day 12 in combined PGCs of both sexes, in female but not male PGCs on day 13, and in both sexes on day 14. c-kit mRNA is undetectable on day 15 in either sex. c-mos is not expressed at detectable levels on day 12 in either sex, but increases gradually in female PGCs to very high levels on day 15. In male PGCs, c-mos is expressed at high levels on days 13–15. Our results are consistent with a role for protooncogenes c-myc, c-fos and c-jun in mediating the initial differentiation of PGCs of both sexes that occurs upon colonization of the gonad. Because c-kit and c-mos are expressed differentially in male and female day 13–15 germ cells, they may play roles in initiating or mediating progress along the sex-specific pathways of development that PGCs embark upon at this time.

Methylation of CpG sites of two X-linked genes coincides with X-inactivation in the female mouse embryo but not in the germ line.

To further our understanding of initiation and imprinting of X-chromosome inactivation, we have examined methylation of specific CpG sites of X-linked Pgk-1 and G6pd genes throughout female mouse development. Methylation occurs around the time of inactivation and earlier for Pgk-1, which is closer to the X-inactivation centre. In female primordial germ cells, the inactive X chromosome escapes methylation; this may underly the reversibility of inactivation at meiosis. Similarly, the genes are unmethylated on the inactive X chromosome in sperm; hence, the imprint specifying preferential X-inactivation in extra-embryonic tissues must reside elsewhere.

A family of octamer-specific proteins present during mouse embryogenesis: evidence for germline-specific expression of an Oct factor.

We have analysed various adult organs and different developmental stages of mouse embryos for the presence of octamer-binding proteins. A variety of new octamer-binding proteins were identified in addition to the previously described Oct1 and Oct2. Oct1 is ubiquitously present in murine tissues, in agreement with cell culture data. Although Oct2 has been described as a B-cell-specific protein, similar complexes were also found with extracts from brain, kidney, embryo and sperm. In embryo and brain at least two other proteins, Oct3 and Oct7, are present. A new microextraction procedure allowed the detection of two maternally expressed octamer-binding proteins, Oct4 and Oct5. Both proteins are present in unfertilized oocytes and embryonic stem cells, the latter containing an additional protein, Oct6. Whereas Oct4 was not found in sperm or testis, it is expressed in male and female primordial germ cells. Therefore Oct4 expression is specific for the female germline at later stages of germ cell development. Our results indicate that a family of octamer-binding proteins is present during mouse development and is differentially expressed during early embryogenesis. Protease clipping experiments of Oct4 and Oct1 suggest that both proteins contain similar DNA-binding domains.

Cosexuality in the Leech,Nephelopsis obscura(Erpobdellidae)

Summary Hermaphroditism of the erpobdellid leach Nephelopsis obscura is described on the basis of macroscopic and histological studies of the gonads. Perivitellogenic cells (female primordial germ cells) proliferate from the septa of segments XII and XIII in individuals of 26–30 mg and spermatogenic cells from segments XII to XIII of 20–25 mg specimens. The majority of hatch- lings develop functional testisacs when they reach 170 mg and functional ovisacs when they reach 250 mg. Oocytes proliferate only after the formation of stage 5–6 spermatozoa. Thus, N. obscura is a sequential cosexual hermaphrodite with spermatogenesis and mature spermatozoa development commencing before oogenesis. During the second cycle of gametogenesis observed in larger animals, both mature spermatozoa and ova co-occur and N. obscura exhibits simultaneous hermaphroditism.

Synthesis of glycoconjugates in mouse primordial germ cells

The synthesis of protein-bound carbohydrates has been studied in primordial germ cells (PGCs) and in somatic cells of 12.5 to 13.5-days-postcoitum (dpc) fetal mouse gonads. Both cell types were shown to synthesize asparagine-linked glycopeptides and glycosaminoglycans (GAGs). In addition, PGCs also synthesize lactosaminoglycans (LAGs) although in different proportions in female and male germ cells. Female PGCs, which at 13.5 dpc are entering meiosis, synthesize mainly LAGs, and minor amounts of hyaluronic acid (HA) and chondroitin sulfate (CS). Male germ cells, on the other hand, synthesize mainly CS. Furthermore, somatic cells of fetal gonads synthesize HA as the major class of GAGs. It is suggested that the activation of LAG synthesis in developing germ cells might be related to the beginning of meiosis. Moreover, we propose that HA synthesis might be developmentally regulated in somatic cells of the gonad, in order to regulate the establishment of specific interactions with germ cells.

Adhesiveness of mouse primordial germ cells to follicular and Sertoli cell monolayers

The adhesiveness of female and male mouse primordial germ cells (PGCs) to somatic cell monolayers of various origin has been studied in the definite conditions of an in vitro system. PGCs were isolated from the gonads of embryos of various post coital ages according to the method of De Felici & McLaren (1982), and seeded on the cell monolayers. PGCs from 12.5 to 15.5 days post coitum (dpc) embryos specifically adhered to Sertoli and follicular cells obtained from adult gonads. The percentage of female PGCs which adhered to follicular cell monolayers was significantly higher than that of male PGCs. No significant adhesion was seen between PGCs and somatic cell monolayers obtained from various embryonic and adult tissues. The results obtained indicate that the simple in vitro assay described in the present paper might help to characterize the cellular interactions between somatic and germ cells during gonadal development.

Random X-chromosome inactivation in female primordial germ cells in the mouse

The pattern of expression of the two X chromosomes was investigated in pre-meiotic germ cells from 12 1/2-day-old female embryos heterozygous for the variant electrophoretic forms of the X-linked enzyme phosphoglycerate kinase (PGK-1). If such germ cells carry the preferentially active Searle's translocated X chromosome (Lyon, Searle, Ford & Ohno, 1964), then only the Pgk-1 allele on this chromosome is expressed. This confirms Johnston's evidence (1979, 1981) that Pgk-1 expression reflects a single active X chromosome at this time. Extracts of 12 1/2-day germ cells from heterozygous females carrying two normal X chromosomes show both the A and the B forms of PGK; since only one X chromosome in each cell is active, different alleles must be expressed in different cells, suggesting that X-chromosome inactivation is normally random in the germ line. This result makes it unlikely that germ cells are derived from the yolk-sac endoderm where the paternally derived X chromosome is preferentially inactivated. In their pattern of X-chromosome inactivation, germ cells evidently resemble other tissues derived from the epiblast.