Lab 1

Gametogenesis

(Adapted from "A Laboratory Manual of Comparative Anatomy and Embryology" by Florence Moog and Marilyn Krukowski)

Introduction The production of gametes in animals of all kinds involves the transformation of what appear to be ordinary epithelial cells into highly differentiated sex cells, each prepared to mate with a cell of opposite type to form a new individual. The essential event in this conversion, in both males and females, is a reduction in the amount of chromatin present, so that the finished gamete contains only half as many chromosomes as all other cells of the body. This reduction is effected by two divisions differing from ordinary mitotic divisions, and known as meiotic or maturation divisions.

In males meiosis occurs when the future sperm still resembles a generalized somatic cell. The reduction of chromosome number is therefore followed by reduction of cytoplasm and condensation of chromatin, so that the sperm becomes an extremely small, compact, motile body. Only after this metamorphosis has been accomplished is the sperm able to fertilize eggs. In females, on the contrary, completion of meiosis is preceded by a period of growth or yolk accumulation, since even the smallest eggs are considerably larger than somatic cells. Ova are therefore able to mature and become fertilizable at the same time.

Although the processes of gamete formation and gamete union differ in detail among different animals, they are in their essential features uniform throughout the animal kingdom. We will study oogenesis in the mammal (cat or rabbit) and spermatogenesis in the grasshopper.

I. The Formation of Ova

A. Oogenesis in the Mammal

The first important difference between egg and sperm formation lies in the period of growth and yolk accumulation, which the future ovum undergoes before maturing. The processes of growth can be readily studied in the adult mammalian ovary, in which eggs in all stages of development may be observed. Examine a slide showing a cross section through the ovary of a cat or other small mammal.

Surveying the slide as a whole under low magnification, distinguish between the cortex and the medulla. The medulla consists mainly of loose connective tissue and numerous contorted blood vessels; the cortex is conspicuous for the great number of oocytes it contains. Note that these germ cells have prominent nuclei.

The ovary is covered by a continuous sheet of epithelium customarily designated as the germinal epithelium. This layer was so named in the belief that it gives rise to the future oocytes. In spite of our current knowledge which clearly indicates that primordial germ cells have an extragonadal origin and that these cells migrate to and enter the substance of the developing ovary early in embryonic life, the term germinal epithelium with its erroneous and misleading implication, persists in the literature. The germinal epithelium consists of ovarian tissue, not future egg cells.

Immediately beneath the epithelium is a dense layer of connective tissue, the tunica albuginea and below this, embedded in the cortex, are clusters of immature egg cells or primordial follicles. Each primordial follicle consists of a large round primary oocyte surrounded by a single layer of flattened follicular or granulosa cells (not always easily discernible). Maturation of the follicle begins when an immature egg cell together with its surrounding layer of follicle cells leaves the egg cluster and moves deeper into the cortex. The oocyte enlarges and the associated flattened follicular cells become cuboidal or low columnar in appearance. This is the earliest stage of the primary follicle. The mitotic division of the follicular cells results in a stratified epithelium, henceforth designated as the granulosa layer or stratum granulosum. Between the oocyte and the granulosum, one can sometimes see a refractile, deeply staining layer, the zona pellucida. These structures are illustrated in Figure 1 and Figure 2. [The figures for this lab were scanned from Atlas of Descriptive Embryology, 4th ed by Willis W. Matthews, Macmillan Publishing Co. 1986]

As the follicles continue to grow they move still deeper into the cortex. The granulosa cell layer continues to proliferate and concurrently, a sheath of stromal or connective tissue cells, the theca, develops around the follicle external to the follicle cells. In time the theca differentiates into two distinct layers: the theca interna, a vascular inner layer of estrogen-producing secretory cells and the theca externa composed primarily of connective tissue.

Further development of the follicle is associated with the appearance of irregular spaces, filled with liquor folliculi, in the stratum granulosum. The irregular spaces become confluent forming a large fluid-filled cavity known as the follicular cavity or antrum. (Precipitation of protein in the fluid may give the cavity a granular appearance.) The antrum is lined by the stratum granulosum which in one region is thickened and projecting into the antral cavity. The thickened region, the cumulus oophorus, has the ooctye in its center. Thus the developing egg cell, which initially had a central position in the follicle, is now eccentrically located. At this stage of development the follicle is designated as a secondary or Graafian follicle.

At the time when formation of the antrum begins, the oocyte has usually reached its full size. Although the ovum undergoes no further growth, the follicle as a whole continues to enlarge and when of maximum size it is a large vesicle, which bulges from the free surface of the ovary.

Find a primary follicle in which the oocyte is surrounded by a single layer of epithelial follicle cells. Then find older follicles in which the number of epithelial cells has increased, and still older ones in which spaces have begun to appear among the follicle cells. Are there follicles containing more than one ovum?

(NB: The histological stains used in the two ovary slides color nuclei bluish; cytoplasm and liquid secretions containing protein are colored red or pink. Chromatin is the material of which the chromosomes are built. It is largely DNA combined with histones. It derives its name from the fact that it has a high affinity for certain dyes used in cytological preparations, and thus becomes intensely colored, or chromatic.)

Locate a mature follicle and identify the cumulus, oocyte, follicular cavity, and the stratum granulosum, surrounded by the theca interna and theca externa. At ovulation, the follicular wall will burst open and the ovum, together with its surrounding cumulus cells and liquid contents of the follicular cavity, will be cast out into the oviduct or Fallopian tube.

Optional Exercise: Measure the diameter of an ovum in a mature follicle using an eyepiece reticule (see Measuring with the Microscope ). Compare its size to a young oocyte. How much would you estimate that its volume has increased during oogenesis?

Among mammals generally the egg completes its first meiotic division in the Graafian follicle shortly before ovulation and then pauses in the metaphase of the second meiotic division. The second division is completed in the Fallopian tube, after the sperm has fertilized the egg. It is of interest that not all the follicles that can be found in the ovary reach the point of expelling ripe eggs. Most of them break down and are resorbed at some stage of their development; many of the primordial follicles never develop any further.

B. Corpus luteum formation

Upon expulsion of the ripe ovum and the liquid that fills the antrum or follicular cavity, the Graafian follicle collapses. Quickly, however (within a few days in the human), the follicle begins to expand again because of the enlargement of the epithelial follicular cells. These cells accumulate a variety of lipid-rich substances including, in most species, a yellow pigment called lutein. At the same time some of the theca cells spread into the interior, producing connective tissue fibers that ramify through the body and form a loose connective tissue core at the center. Numerous blood vessels also penetrate the mass. Very soon the corpus luteum begins to function as an endocrine organ, producing progesterone, which promotes the development of the compact layer on the surface of the uterine endometrium.

If the liberated egg has not been fertilized, the luteal body formed within its follicle shortly disintegrates; in the human it survives only during the two weeks from ovulation to menstruation. If fertilization does occur, however, the corpus luteum persists and continues to grow for a part of the pregnant period. During this time it constitutes an indispensable source of progesterone. Later it begins to undergo involution, and some time after delivery is finally reduced to a retracted scar on the surface of the ovary.

Examine a corpus luteum in a section of the ovary of a pregnant mammal; such a corpus is often referred to as a "true corpus luteum," or "corpus luteum of pregnancy," to distinguish it from the smaller and less-developed structure which is found when pregnancy does not occur. Under low power note the size of the corpus relative to the size of the ovary. Identify the connective tissue sheath, the theca which surrounds the body. Under higher power examine the large lutein cells which make up the bulk of the structure. Observe the numerous blood vessels and connective tissue fibers. The central connective tissue mass will not appear in all sections. Why would this be so?

II. The Formation of Sperm

A. Spermatogenesis in the Grasshopper

1. Spermatogonia to spermatids. The animal used is the large Florida grasshopper, Rhomelia. The testis of this form is made up of a number of long narrow lobes, which are arranged with their blunt ends facing the surface of the organ and the more pointed ends directed toward the center. In any preparation some lobes will have been cut across, some obliquely, and some almost parallel to the long axis, so that all regions of the lobe are in a continuous piece. Focus your attention on a section of the latter type. Note that the lobe is made up of many separate compartments or cysts. Each cyst contains potential sperm cells in the same stage of development. As development proceeds the cyst is pushed toward the inner end of the lobe, and more immature cysts take its place at the periphery. [NB. The slide was stained using the Feulgen method that is specific for DNA. This stain is useful for studying chromosome and nuclear structure. The cytoplasmic components are colored yellow or brown.]

At the outer edge of the lobe are the spermatogonia. These cells multiply for some time before beginning to undergo maturation. They have large nuclei containing conspicuous masses of chromatin, and occasionally mitotic division figures may be seen. A spermatogonium that has replicated each chromosome in preparation for the first maturation or meiotic division and entered meiotic prophase I is called a primary spermatocyte.

Primary spermatocytes are found in cysts internal to the spermatogonia. Study cysts of primary under high power (100X, oil immersion), but be careful to avoid crushing the slide. The youngest cysts contain cells with large nuclei in which the chromosomes are seen as long thin threads. Each of the threads is called a chromatid. [Remember that each replicated chromosome consists of two chromatids joined together at the centromere.] A dark solid body near the nuclear membrane is the sex (X) chromosome, which assumes a thread-like form later than the others. As the cyst moves down the lobe, homologous pairs of chromosomes come together forming synaptonemal complexes and twist around each other. In this twisting they often break and reform in such a way that pieces are exchanged between the chromatids (crossing over). [Recall that prophase of meiosis I involves pairing of homologous chromosomes. In addition, exchange of pieces of chromatin from different chromosomes via crossing over, brings about additional new combinations of genes. Such exchange does not normally occur in mitosis.] At the same time, as prophase progresses, the threads become shorter and thicker (i.e., more condensed); you can see this change by examining cysts farther down the lobe toward the lumen.

Ultimately the sets of four chromatids align on the metaphase plate and begin to separate into pairs. Because of the way in which the chromatids have coiled about one another, however, they are immediately unable to come apart completely but cling together in curious ring- and cross-shaped structures. These structures are called tetrads. Find a cyst containing very short, club-like chromatids and see whether you can make out groups of four.)

The tetrads finally separate into pairs, and the primary spermatocyte then undergoes its first maturation division to become a pair of secondary spermatocytes. Each secondary spermatocyte contains one pair of sister chromatids from each tetrad. When the condensed chromosomes in secondary spermatocytes align on the metaphase plate, they are known as dyads. Search carefully for a cyst in which the members of each dyad are closely paired or slightly parted from each other, (i.e., the chromosomes are in the metaphase to anaphase transition). After telophase and cytokinesis, the result is the formation of fully matured cells, or spermatids, which are provided with the haploid number of chromosomes.

2. Spermatids to sperm. The spermatids at first resemble ordinary cells; but soon they begin to change into small, compact, tailed bodies. This metamorphosis may be observed in the inner regions of the lobe. First the spermatid elongates, most of the cytoplasm is sloughed off, except for a small amount which becomes the tail sheath surrounding a fine tail filament which grows back from the centrosome. Meanwhile the chromatin mass becomes more condensed and elongated to form the solidly staining head. The definitive sperm, which is ready to fertilize eggs, consists of the head (which in the grasshopper is ten or more times as long as it is wide), the middle piece, containing the centrosome and mitochondria, and the extremely long tail. Can you see sperm tails in this slide?

Optional Exercise: Using your eyepiece reticule and a calibrated stage micrometer, measure the diameter of the head of a sperm. You will need to do this using your oil immersion (100X) objective. What is the maximum head length you can measure?

This page was posted 25 January 2003

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