Analysis of Biological Development (K. Kalthoff)

Updates to Topic 03: Gametogenesis


Answers to Questions in Text

RNA transport into Fly Oocyte (p. 62-64)

  1. In autoradiographs like the one shown in Fig. 3.17a, the radiolabel over the oocyte nucleus is always spurious, even when the nucleus is clearly in the plane of section. What does this mean in terms of rate of RNA synthesis in the oocyte? Answer: The rate of RNA synthesis in the oocyte itself is small in comparison to that of the nurse cells and follicle cells, both of which are polyploid.
  2. The method of autoradiography registers the presence of the radioisotope used, regardless of the molecule in which it is incorporated. The radioisotope used in this experiment was tritium (3H), which has the chemical properties of hydrogen and enters the same metabolic pathways as hydrogen. The investigators therefore needed a way of testing whether the radioactivity they saw on their autoradiographs was still present in RNA, rather than having been metabolized into other hydrogen-containing molecules. What do you think they may have done? (Hint: Their tissue sections were prepared in a way that leaves the tissue accessible to water-soluble agents.) Answer: They incubated some of their sections with RNase before autoradiography. This treatment removed most of the label seen in the untreated slides, indicating that most of the radiolabel was present in RNase-sensitive molecules, most likely, in RNA.
  3. Do the autoradiographs show what type(s) of RNA are transferred from nurse cells to oocyte? If not, what additional experiment could the investigators do to find out? See Method 3.1. Answer: In follow-up experiments, they extracted total RNA from oocytes, separated it according to size by gel electrophoresis, and determined the size and abundance of the radiolabeled RNA classes by autoradiography. Most of the RNA was ribosomal.

Vitellogenesis (p. 65)

  1. The process of receptor-mediated endocytosis was not well understood when Telfer did the experiment described here. Which of his observations were strongly indicative of the involvement of a specific receptor in the uptake of the female-specific protein into oocytes? Answer: The fact that female-specific protein - but not other proteins - accumulated in the recipient's oocytes until its concentration was 20 times greater in the eggs than in the hemolymph.
  2. Working to Telfer's advantage was the fact that vitellogenin from Hyalophora cecropia matches the vitellogenin receptor of Antherea polyphemus well enough to be taken up into the oocyte. If this had not been the case, what alternate experimental design could Telfer have used? Answer: He could have pulse-labeled all proteins synthesized in the moth by injecting them with a radiolabeled amino acid. After allowing the injected moths to survive for increasing periods of time, he could have isolated ovaries, other organs, and hemolymph from females and analysed newly synthesized proteins in each by gel electrophoresis. He would have found large amounts of radiolabeled female-specific protein first in fat body, then in hemolymph, and finally in oocytes.
  3. Given the advantages of synthesizing yolk proteins in a large organ outside the oocyte, what may be the adaptive value of using a specific family of storage proteins as opposed to withdrawing a fraction of all hemolymph proteins into oocytes? Answer: The synthesis of specific proteins is easy to control through the transcription of the few genes that encode them. The activation of these genes can then be made sex-specific (only in females) and dependent on a female's stage in her life cycle, her nutritional state, etc. Also, one type of receptor will suffice for receptor-mediated endocytosis of one or a few similar proteins.

Comments

See Movies on Oogenesis on Movies page.

Clarifications and Corrections

New Review Articles

Greenfeld C. and Flaws J.A. (2004) Renewed debate over postnatal oogenesis in the mammalian ovary. BioEssays 26: 829-832
Hecht N.B. (1998) Molecular mechanisms of male germ cell differentiation. Bioessays 20: 555-561
Matowa N. and CooleyL. (2001) Comparative aspects of animal oogenesis. Devel. Biol. 231: 291-320
Matzuk M.M., Burns K.H., Viveiros M.M. and Eppig J.J. (2002) Intercellular communication in the mammalian ovary: Oocytes carry the conversation. Science 296: 2178-2180
Sardet C., Prodon F., Dumollard R., Chang P. and Chenevert J. (2001) Structure and function of the egg cortex from oogenesis through fertilization. Devel. Biol. 241: 1-23

New Research Articles

Johnson J., Canning J., Kaneko T., Pru J.K. and Tilly J.L. (2004) Germ line stem cells and follicular renewal in the postnatal mammalian ovary. Nature 428: 145-150
Eggan K., Jurga S., Gosden R., Min I.M. and Wagers A.J. (2006) Ovulated oocytes in adult mice derive from non-circulating germ cells. Nature 441: 1109-1114

Johnson et al. propose that mammalian ovaries may be seeded continually by circulating germ cells derived from bone marrow. Eggan et al. find no evidence that any circulating cells give rise to mature oocytes, thus supporting the traditional view that female mammals produce a limited supply of oocytes prenatally.
Kiger A.A., White-Cooper H. and Fuller M.T. (2000) Somatic support cells restrict germline stem cell self-renewal and promote differentiation. Nature 407: 750-754
Kiger A.A., Jones D.L., Schulz C., Rogers M.B. and Fuller M.T. (2001) Stem cell self-renewal specified by JAK-STAT activation in response to a support cell cue. Science 294: 2542-2545
Tran J., Brenner T.J. and DiNardo S. (2000) Somatic control over the stem cell lineage during Drosophila spermatogenesis. Nature 407: 754-757
Tulina N. and Matunis E. (2001) Control of stem cell self-renewal in Drosophila spermatogenesis by JAK-STAT signaling. Science 294: 2546-2549

A fundamental characteristic of stem cells is their capacity to produce daughter cells that either retain stem cell identity or initiate differentiation. The dual progeny may originate simultaneously by asymmetrical division, as indicated in Figure 3.11 of the text, or by alternating stochastically between producing daughter cells that are either stem cells or committed progenitors. The four studies quoted above are beginning to elucidate the signaling pathways that govern this balance in the Drosophila testis. Here, germ line and somatic stem cells attach to a cluster of support cells called the hub (see Fig. 1 below). Both Kiger et al. (2001) and Tulina/Matsunis find that the hub specifically expresses Unpaired, a ligand that activates a Janus kinase (JAK), which phosphorylates a signal transducer and activator of transcription (STAT). They also show that in the absence of JAK-STAT signaling germ line stem cells differentiate but do not self-renew. Conversely, ectopic JAK-STAT signaling greatly expands both germ line and somatic stem cell populations. These results indicate that the hub cells signal to adjacent stem cells through the JAK-STAT pathway, thereby promoting stem cell renewal. A competing signal - one that promotes stem cell differentiation - appears to be delivered by other somatic support cells including cyst cells, which surround the developing germ cells as they move away from the hub. In a previous study, Kiger et al. (2000) found that these cells restrict stem cell renewal. Loss of function of epidermal growth factor receptor in these cells causes an increase in the number of germline stem cells. Similarly, Tran et al. observed that testes lacking Raf activity in somatic cells have excess stem cells, which remain active longer than in wild type. Together, the data indicate that the Unpaired signal from the hub defines a niche for stem cell renewal, and that stem cell daughters displaced from the hub come under the influence of competing signals from other support cells that initiate differentiation in the stem cell lineages.

Fig.1 Apex of a Drosophila Testis. (A) Germ line stem cells (five to nine cells, yellow) can divide asymmetrically. Their daughters remain stem cells as long as they are attached to a cluster of about 12 somatic hub cells (red). In contrast, daughter cells displaced from the hub develop into gonialblasts (blue). These undergo four rounds of mitosis with incomplete cytokinesis, forming 16 interconnected spermatogonia (pale blue). Likewise, somatic stem cells (gray) divide to form new stem cells or cyst cells that envelope the spermatogonia. (B) Unpaired mRNA produced in the hub cells is made visible (dark blue stain) by in situ hybridization. (C) Unpaired protein in the hub cells is made visible (blue stain) through an insert with -galactosidase activity. The Unpaired protein initiates the JAK-STAT signaling pathway, which promotes stem cell renewal. From Tulina and Matunis (2001).

Van Doren M., Tarczy Brihier H., Moore L.A. and Lehmann R. (1998) HMG-CoA reductase guides migrating primordial germ cells. Nature 396: 466-469

Starz-Gaiano M., Cho N.K., Forbes A. and Lehmann R. (2001) Spatially restricted activity of a Drosophila lipid phosphatase guides migrating germ cells. Development 128: 983-991

The migration of primordial germ cells exemplifies the general ability of many cells to receive directional signals from their environment and to translate the signals into cytoskeletal rearrangements that cause directional movement. The two papers quoted above harness the power of Drosophila genetics to define some of the molecular pathways that generate germ cell attractants and germ cell repellents.

Van Doren et al. have identified a gene, named columbus+, which is highly expressed in the mesodermal gonad rudiments while it is being populated by migrating primordial germ cells. Misexpression of columbus+ in other tissues, such as the nervous system, is sufficient to divert primordial germ cells to those tissues. The columbus protein has 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) activity, and the catalytic domain of the enzyme is necessary for is function in germ cell attraction. The natural substrate of HMG-CoA in this function remains to be elucidated.

Starz-Gajano et al. have identified two genes, wunen+ and wunen-2+, which are redundantly expressed in areas of the gut that are avoided by germ cells while they are migrating to the gonad rudiments. Misexpression of wunen+ or wunen-2+ in other tissues causes migrating germ cells to avoid these tissues as well and also reduces the number of surviving germ cells. The proteins encoded wunen+ or wunen-2+ have a phosphatidic acid phosphatase activity, and the catalytic domains of the proteins are necessary for their germ cell-repelling function. The authors propose that the wunen activity may be involved in generating a repellent or destroying an attractant of germ cells.


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