Download Morphology of Cricket Ovaries Role of Microtubules and

Morphology of Cricket Ovaries
Role of Microtubules and Microfilaments in Endocytosis
I.
Microscopic Observation of Cricket Ovary
During the lab in which you prepared your electrophoresis samples, you were also able to
observe ovaries that had been removed from an adult female cricket. You should have seen:
- mature chorionated eggs that had been released from the ovarioles,
- small portions of ovary with attached ovarioles, and
- ovarioles containing previtellogenic and vitellogenic oocytes.
Each ovariole is a chain of sequentially developing oocytes enclosed within an epithelial
sheath called the ovariole sheath. The nucleus of each small, immature, non-vitellogenic
oocyte is visible in the center of the clear cytoplasm. Vitellogenic oocytes are larger and
have a milky-white cytoplasm due to the presence of the yolk proteins that are being
sequestered and are accumulating in yolk spheres. Fully mature eggs appear glossy and
yellowish-white due to the water-proof chorion surrounding the egg.
The follicular epithelium, a single layer of small cells surrounding the oocyte, is
especially visible around early vitellogenic oocytes.
II. Procedure
A. Observation of Ovariole and Oocyte Morphology
1. Obtain watch glasses or Petri dishes containing ovaries from an adult female cricket and
an immature cricket.
2. Tease away a group of 3-4 ovarioles from the adult ovaries and observe them against a
dark background using a dissecting microscope. Find the individual oocytes. Aside from
being different sizes, do they all look the same? Based on your study of insect ovarian
morphology in Tutorial I and in Tutorial V, identify the vitellogenic and non-vitellogenic
oocytes.
3. Examine ovarioles from an immature cricket in the same manner. How are they different
from the adult ovarioles?
4. Can you find the cloud-like structures on one or both sides of the germinal vesicle
(nucleus) of a nonvitellogenic oocyte? These structures called Balbiani bodies (nuage
bodies) and have recently been found by Dr. James Bradley to be composed of dense
clusters of mitochondria and associated microtubules in cricket oocytes.
B. Observation of Endocytosis
1. Use the dissecting microscope to examine ovarioles from adult female and immature
female crickets that were injected with 0.05 ml of a 1% trypan blue solution and dissected
after four hours.
2. Have any of the oocytes sequestered the dye? If so, is there a relationship between Ablue@
oocytes and vitellogenic oocytes? Compare these ovarioles with those from crickets that
were not injected with dye.
C. Role of Microtubules and Microfilaments in Endocytosis
Use the dissecting scope to examine oocytes and compare endocytosis in oocytes in each
of four treatment groups. Labeled dishes contain ovaries of crickets injected with 0.05 ml of a
1% solution of trypan blue 30 minutes after being injected with the drugs listed below. The
four treatments are:
C
C
C
C
Control--Sexually mature, adult females treated with cricket saline (use ovaries
from part B).
Control--Sexually immature, last nymphal instar females pretreated with cricket
saline (from part B).
Mature females treated with cytochalasin B (a drug that inhibits the normal
functioning of microfilaments ) prior to dye injection.
Mature females pretreated with colchicine (a microtubule inhibitor) prior to dye
injection
Describe your observations and address the following questions:
1.
2.
3.
4.
5.
Do fully mature (chorionated) eggs sequester the dye?
Do all of the immature oocytes in an ovariole from a mature female sequester the dye?
Do immature females possess pinocytotically active oocytes?
Are microtubules involved in pinocytotic activity?
Are microfilaments involved in pinocytotic activity?
It is believed that in most insects the same hormone (juvenile hormone) is responsible for
both the induction of vitellogenin synthesis in the fat body cells and the subsequent VG
uptake by vitellogenic oocytes. The way in which juvenile hormone stimulates vitellogenin
uptake is not understood, but one suggestion that has been made is that oocytes must reach
a certain stage of development within the ovariole before they are able to respond to the
presence of juvenile hormone. Do your observations provide any evidence for or against this
suggestion?
In your discussion section, propose an experiment that would test the idea that the
absence of pinocytotically active oocytes in immature females may not be due to the absence
of a hormone in these animals, but simply due to the absence of vitellogenins in the
hemolymph. In other words, maybe the presence of vitellogenins alone is sufficient to induce
pinocytotic activity.
Pre-Lab Quiz
Tutorial V
Morphology of Cricket Ovaries
Role of Microtubules and Microfilaments in Endocytosis
Name____________________________
Lab Day/Table_______________
1. How do vitellogenins (VGs) enter oocytes?
2. What is the source of the VGs in oocytes?
3. What are three stages of cricket oocytes?
4. What dye can be taken up by oocytes Ain vivo?@
5. What is an ovariole?
6. What are the components of Balbiani bodies?
7. Name a drug that inhibits microtubule function.
8. Name a drug that inhibits microfilament function.