Folliculogenesis and oogenesis are important events preceding the release of the mature oocyte (ovulation), as well as for in vitro oocyte maturation, which is. Drosophila oogenesis is an excellent system for the study of developmental cell biology. Active areas of research include stem cell. Spermatogenesis and Oogenesis. Pgs. (Campbell). Spermatogenesis. Production of sperm. Continuous and prolific; Each ejaculation.
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Drosophila oogenesis is an excellent system for the study of developmental cell biology. Active areas of research include stem cell maintenance, gamete development, pattern formation, cytoskeletal regulation, intercellular communication, intercellular transport, cell polarity, cell migration, cell death, morphogenesis, cell cycle control, and many more.
Methods for studying oogenesis
The large size and relatively simple organization of egg chambers make them ideally suited for microscopy of both living and fixed whole mount tissue.
A wide range of tools is available for oogenesis research. Newly available shRNA transgenic lines provide an alternative to classic loss-of-function F2 screens and clonal screens. Protein trap lines provide fluorescent tags oogeneis proteins expressed at endogenous levels for live imaging and screening backgrounds.
This review provides information on many available reagents and key methods for research in oogenesis. Oigenesis in Drosophila [ 12 ] supports an impressively high level of fecundity.
Movement of egg chambers oogenrsis facilitated by peristaltic contractions of circular muscles surrounding each ovariole and a muscle mesh surrounding the whole ovary [ 3 — 5 ].
The ovarioles of each ovary jurnxl at a lateral oviduct, which connects to a common oviduct and then the uterus. Mature stage 14 eggs move into the uterus where they are fertilized by a single sperm and then laid. The development of each egg takes about eight days: Each of the ovarioles produces approximately two eggs per day, or over 60 eggs from a young, well-fed female [ 6 ].
Methods for studying oogenesis
This high output of eggs depends on an abundant source of food, major contributions from support cells in egg chambers and a wide range of cellular interactions.
An overview of oogenesis and examples of how its study has contributed to our understanding of developmental biology are summarized in this section. Oogenesis begins in the germarium center boxwhere germline and pogenesis stem cells GSC and FSC, respectively divide continuously to support the formation of new egg chambers. See text for further details.
The formation of egg chambers also called follicles takes place during the first four days of oogenesis in the germarium.
Germline stem cells at the apical end of germaria are maintained by signaling from adjacent niche cells called cap cells.
Stem cell daughters called cystoblasts leave the niche and undergo four mitotic divisions to produce a cyst of 16 cells. Incomplete cytokinesis in these divisions leaves intercellular bridges that are later stabilized by the accumulation of filamentous actin to form ring canals that persist ujrnal the end of oogenesis. Somatic escort cells encase the dividing cysts. After completing mitosis, escort cells are exchanged for follicle cells to complete stage 1 egg chamber assembly.
The follicle cells are generated by two follicle-cell stem cells located between germarium regions 2a and 2b. Thus, the germarium contains two types of stem cells maintained in separate niches whose division rates must be coordinated to produce cells necessary for egg chamber production.
Only one of the 16 germline-derived cells develops into an oocyte while the remaining 15 differentiate into nurse cells with polyploid nuclei. By the time an egg chamber emerges from the germarium, the oocyte is already positioned at the posterior as a result of a signaling cascade emanating from the next most mature egg chamber. Examination of egg chamber assembly carried out in many laboratories has yielded a huge amount of information on stem cell and stem cell niche biology [ 78 jurbal and the origins of polarity [ 9 ].
This research takes advantage of powerful genetic and cytological tools developed over many years. Most of this time is needed for previtellogenic egg chamber jurna during stages 2—8, when oocyte growth is mediated entirely by intercellular movement of cytoplasm from nurse cells to the okgenesis through ring canals.
The diameter of ring canals slowly expands as egg chambers grow, mediated by active actin filament polymerization in the ring canal rim [ 10 ]. The polarization of oocytes is established during these stages as a consequence of the posterior location of judnal oocyte nucleus. The gurken mRNA accumulates at the posterior with the oocyte nucleus, and produces a ligand for EGFR activation in posterior follicle cells [ 11 ]. In addition, many maternal mRNAs and RNA-binding proteins accumulate specifically in oocytes through a combination of microtubule-based directed transport and trapping within the oocyte [ 12 ].
Follicle cells also make crucial contributions to previtellogenic oogenesis. Egg chambers change shape from spherical to elongated starting during stage 5.
Interestingly, this shape change is driven by egg chamber rotations that occur between stages 5 and 9. Follicle cells drive the rotations as they migrate perpendicular to the axis of the ovariole, laying down a girdle of polarized extracellular matrix as they go. As a result, expansion of egg chambers takes place anisotropically toward the poles [ 13 ]. A major change in cell cycle also takes place in follicle cells. Mitotic divisions cease at the end of stage six, followed by three endoreplication cycles during stages 7—10A [ 14 ].
The Notch-Delta signaling pathway controls this transition [ 1516 ], providing an excellent opportunity for studying cell cycle changes under developmental control. These ring canals do not grow in size during oogenesis, but they are able to support intercellular movement of protein between cells, raising the interesting possibility that they serve an important function in oogenesis [ 2021 ].
Stage 8 egg chambers do not progress into vitellogenesis yolk uptake if egg chambers have severe patterning defects or if environmental conditions are unlikely to support the survival of progeny. During egg chamber apoptosis, follicle cells lose oovenesis epithelial organization and become phagocytic, engulfing the cytoplasm of germline cells [ 2223 ].
If egg chambers are sound and protein is restored to the food, oogenesis resumes and stage 14 eggs can develop from surviving stage 8 egg chambers within one day. Thus, stage 8 serves as a metabolic checkpoint that triggers egg chamber destruction while preserving less mature egg chambers poised to resume development when conditions improve. The final day of oogenesis produces a huge increase in oocyte volume due to yolk uptake from hemolymph and the complete transfer of nurse cell cytoplasm to the oocyte.
Yolk uptake beginning pogenesis the end oogenesie stage 8 causes the rate oocyte growth to overtake nurse cell growth so that the oocyte takes up half the volume of egg chambers by stage 10A. During these stages, several key patterning molecules are localized within the oocyte: The movement and anchoring of these maternal mRNAs are active areas of research that benefit from the ability to do live-cell imaging to reveal conserved mechanisms of mRNA localization [ 2526 ].
Nurse cell death after dumping has some of the hallmarks of apoptosis, although it is a caspase-independent process [ 2327 ]. In preparation for this nurse cell dumping, stage 10B egg chambers produce cables of unipolar actin filaments that grow from the nurse cell membranes inward until they oogenesiw the nuclear envelope [ 2829 ].
The actin cables prevent nurse cell nuclei from being squeezed into ring canals where they would block the flow of cytoplasm to the oocyte.
Prostaglandin signaling is involved with triggering the formation of nurse cell actin cables [ 30 ]. They secrete yolk protein during vitellogenesis, vitelline membrane proteins during stage 9—11, and finally chorion proteins beginning in stage In preparation for chorion protein production, stage 9 follicle cells switch from endoreplication to synchronous amplification of the two chorion-gene clusters through repeated firings of replication origins in the clusters [ 3132 oogenesia.
The endocycle to amplification switch is mediated by down-regulation of Notch and activation of the Ecdysone receptor [ 33 ]. In addition to driving egg chamber rotation, follicle cell migrations dramatically change egg chamber organization. During stage 9, 6—10 border cells delaminate from the anterior follicle cell epithelium and migrate between nurse cells to the anterior of the oocyte carrying two polar cells as passengers.
The migration of these cells is guided by the PVR growth factor produced in the oocyte [ 34 ], and propelled by acto-myosin dynamics [ 35 ].
As border cell migrate between nurse cells, other follicle cells move around nurse cells toward the oocyte where they form a columnar jural. During stage 10A, follicle cells at the anterior circumference of the oocyte migrate centripetally, separating the oocyte from nurse cells. Nurse cell dumping is completed just as centripetal follicle cells completely cover the oocyte anterior at the end of stage The border cells iurnal in forming the micropyle through which a sperm reaches the oocyte.
Two groups of 65—80 follicle cells in the anterior, dorsal domain form specialized eggshell structures called dorsal appendages that facilitate gas exchange during embryogenesis. The specification and morphogenesis of dorsal appendages are mediated by patterning cues in specific follicle cell groups oogenesiw 36 ]. However, using the right UAS vector is critical.
The original UASt vectors include P element ends for transformation and work very well in the somatic cells of the ovary; however, UASt-mediated expression is extremely poor in germline cells. She produced new UASp vectors that instead use the basal promoter from the P element transposase gene. UASp vectors are included in the T. A version of UASp that includes the attB sequence for phiCmediated integration was reported recently [ 38 ].
New UAS, attB vectors made in the Rubin lab at Janelia Farm are optimized for expression in the nervous system [ 39 ]; however, they work very well in follicle cells and also support expression in germline cells. Rubin lab vectors are listed on their website and are available at Addgene. A multitude of Gal4 lines is available from stock centers and labs that are useful for driving expression in specific cells of the ovary. The best source for comprehensive general information is the Gal4 page of the Bloomington Drosophila Stock Center BDSCalthough this page has limited information on expression.
Many of these lines are derived from Gal4 enhancer trap screens aimed at identifying lines expressed in during oogenesis and early embryogenesis [ 4041 ]. Commonly used drivers for oogenesis research are listed in Table 1including lines for expression in the stem cell niche germariumgermline cells and follicle cells.
Gal4 expression from the nanos promoter is in germline stem cells, then recedes in young egg chambers and resumes later in oogenesis.
The otu promoter produces more uniform expression in the germarium and egg chambers, but tapers off toward the end of oogenesis. Most of the lines driving expression in somatic follicle cells are also expressed in other tissues of the kogenesis.
An exception is Vm26a-Gal4, which has the promoter from the vitelline membrane 26Aa gene and is specific for follicle cells in stage 10—14 egg chambers [ 36 oogeenesis. A group of follicle cells with one level of reporter expression is often adjacent to another group jurnak a very different level.
Nurse oogenesus can also display different levels of expression [ 43 ]. The explanation for follicle cell mosaicism may involve epigenetic marks inherited by progeny during mitotic proliferation [ 44 ], though the level of variation in expression is somewhat masked by intercellular movement of proteins between follicle cells through ring canals [ 20 ]. The probability of mosaic expression levels should be taken into consideration when interpreting results of an experiment involving Gal4-mediated expression.
Repression of weaker germline Gal4 drivers may be possible; however, but this needs further testing. Several vectors are available with promoters for germline-specific expression, or for ubiquitous expression in egg chambers Table 2.
The ovarian tumor otu promoter produces low to moderate levels of germline expression jurhal oogenesis, and the hsp26 promoter produces moderate levels of expression starting in stage 8 egg chambers. The nanos nos or vasa vas promoter directs expression in primordial germ cells in developing animals, and in the germline cells of the adult germarium.
Expression is low junral early egg chambers, and high in late stages. For ubiquitous expression, the UbiquitinE Ubi-p63E or spaghetti-squash promoter is a good choice. The greatest strength of Drosophila as an experimental system is the ability to use genetic analysis to understand cellular and developmental processes.
Work from many labs during the past decades has resulted in remarkable innovations in the genetic tools available to study the genes and developmental mechanisms controlling oogenesis. We present here the basis for classical genetic approaches, and also summarize more recently developed techniques that rely on transgenic manipulations.
Initial genetic approaches to study oogenesis focused on screens for recessive loss-of-function mutations affecting female fertility. The two broad classes of oogeneeis affecting female fertility are female-sterile fs mutations and maternal-effect lethal mutations mel. By definition, female-sterile mutations cause a defect during oogenesis such that normal eggs are not produced.
In contrast, females bearing maternal-effect lethal mutations are able to produce normal eggs, indicating that oogenesis is able to proceed normally, but these eggs fail to produce viable offspring due oogenesiis the lack of a gene product essential for embryonic development that is normally provided to oogenesls egg during oogenesis.