DEVELOPMENTAL BIOLOGY - 312

GONADS, GAMETES, AND MEIOSIS

During lab today will we compare the gonads and gametes of several vertebrates and invertebrates. The prepared slides we will be examining and their locations are listed below:
Individual Slide Boxes Demo Slide Boxes On Demonstration
Monkey Ovary Starfish Ovary Honey Bee Ovary
Cat Mature Follicle (or Grasshopper Ovary Crayfish Testis
Graafian Follicle) Human Fetal Ovary
Chicken Ovary Human Fetal Testis
Frog Ovary
Ascaris - Maturation
Rat Testis
Grasshopper Testis
Frog Sperm
Chicken Sperm
Human Sperm

INTRODUCTION

In most organisms we recognize two basic cell types: somatic cells and germ cells. Germ cells give rise to gametes (eggs and sperm) whereas somatic cells give rise to all other cell types in the body. Typically, germ cells are characteristically larger than somatic cells and possess an enlarged nucleus called a germinal vesicle. The point in development when germ cells form varies greatly among species, but often occurs during an embryonic stage. For example, in amphibians and insects germ cells are established very early in development while in bryozoans or hydroids germ cells can not be distinguished until near the time of sexual reproduction. The initial germ cells that form are referred to as primordial germ cells. Primordial germ cells usually have to migrate from where they form to the developing gonad (ovary and/or testis). After reaching the gonad primordial germ cells divide mitotically to generate a large population of germ cells. In males germ cells are spermatogonia and in females germ cells are oogonia. Spermatogonia and oogonia are usually stem cells. Stem cells are undifferntiated cells that divide mitotically producing two types of cells. Some stem cell daughter cells will remain as stem cells to keep the cell line alive. Other stem cell daughter cells will become differentiated. It is the stem cell nature of spermatogonia and oogonia that allow organisms over their life times to produce billions of sperm and eggs.

Sperm and eggs represent two highly differentiated cell types that are specialized to create a zygote through fertilization. During gametogenesis, sperm and eggs develop their characteristic structures. Sperm typcially become elongate cells that contain very little cytoplasm and move using at least one flagellum. In contrast, eggs become very large spherical or oval cells, often containing large amounts of yolk and are immotile. One feature of gametogenesis that is common to the production of both sperm and eggs is meiosis, a type of cell division that reduces the number of chromosome sets each daughter cell contains by half. Consequently, mature gametes are haploid.

Your goals in todays laboratory are:

1. Compare the production of sperm in rats and grasshoppers

2. Compare the morphology of sperm from rats, humans, chickens, frogs, grasshoppers, and crayfish.

3. Compare the ovarian and oocyte structure of monkey, cat, chicken, frog, starfish, grasshopper, and honey bee.

4. Identify meiotic stages during oogenesis in Ascaris

SPERMATOGENESIS

The Mature Testis

We will begin our studies examining a section of rat testis. This section, like most of the sections we will look at today, is stained with hematoxylin and eosin; the former is a blue stain that preferentially stains nuclei blue and the latter is a pinkish-orange counter-stain that is concentrated by acidic components of the cell, including the cytoplasm. Observe the section using the low power objective. Note that most of the testis consists of tangles of seminiferous tubules sectioned in all imaginable planes. The testis is sheathed on the outside by a thick, tough capsule of fibrous connective tissue called the tunica albuginea. Internally, thin sheets of connective tissue (that are continuous with the tunica albuginea) subdivide the testis into a number of lobules, each containing several seminiferous tubules. The blocks of tissues sectioned were mostly from the central part of the testis, but all sections do include a piece of the tunica albuginea.

Under low power note that round, oval, or elongated sections through seminiferous tubules fill most of the slide, but that irregularly shaped spaces occur between the tubules. Examine the areas between seminiferous tubules at higher magnification. You should see that the spaces contain a) small blood vessels that are usually filled with small, disk-shaped, red-stained erythrocytes; b) clumps of cells with conspicuous nuclei that contain clumps of blue-stained chromatin; c) a non-cellular "gelatin" or matrix; and d) connective tissue cells with elongated (spindle-shaped) nuclei. The cells with conspicuous nuclei are interstitial (or Leydig) cells. Interstitial cells produce testerone.

Some of the elongated nuclei are closely associated with the outside of each seminiferous tubules; these nuclei belong to myoid cells whose peristaltic contractions help transport sperm suspended in fluid out of the seminiferous tubules to the epididymis. (The sperm are immotile at this time and cilia occur only at limited sites in the duct system.)

Even brief examination of the seminiferous tubules reveals their cytological complexity and the fact that adjacent tubules are quite different from each other in detail. Experts have spent literally years unraveling the complex sequence of changes that occurs in a given seminiferous tubule over time. In rats, this spermatogenic cycle lasts a total of 48 days, but successive cycles start every 12 days so that there are a total of 4 overlapping cycles at every site along the tubule. A reasonable analogy occurs at SLU: a student usually takes four years (freshman, sophomore, junior, senior) to complete his education. As a given student enters his sophomore year, a new freshmen is starting, etc. Eventually, there are always 4 overlapping "classes."

The "wall" of any seminiferous tubule you observe consists of a complex array of cells. If we could remove all the cells of the germ line, the picture would be very simple. One could see that the tubule itself is formed by a single layer of pyramidal Sertoli cells that form a continuous epithelium. On the outside of each tubule is a sheath of myoid cells; locate the spindle-shaped nuclei of such cells in your section. The Sertoli cells of our "germ cell-free" preparation would be quite "holey" due to the numerous inpocketings that push in from the apex, sides and base of each cell. Each of these pockets is the site for a germ line cell. In actual sections, the germ line cells are so abundant that the cytoplasm of the Sertoli cells is virtually undetectable, but we can locate Sertoli cell nuclei. These are pale staining, large vesicles located near the outer margin of the tubule. Often these nuclei are somewhat triangular in shape and frequently they have irregular indentations of the nuclear envelope; characteristically there is a large red nucleolus in each.

Attempting to figure out the events of spermatogenesis by studying random seminiferous tubules is maddening and unproductive. As a point of focus you should locate and study three specific examples:

A. A tubule in which nearly mature spermatozoa are deeply embedded in the Sertoli cell layer, so that their hook-shaped nuclei reach to the level of Sertoli cell nuclei.

B. A tubule in which spermatid nuclei are still quite round, but the condensation of chromatin has begun along one surface.

C. A tubule in which meiosis is occuring in some of the cells.

There are clues to finding appropriate examples of such stages. Your job will be to find examples on your slide.

Spermatogonia -- really what we are looking for is their nuclei -- can be identified with great confidence using the combination of the following three criteria: 1. spermatogonial cells are restricted to the basal compartment, so their nuclei will be at or below the level of Sertoli cell nuclei; 2. their nuclei tend to be quite small, comparable in size to those of spermatids; and 3. because of their rapid mitotic activity, the nuclei are usually quite darkly staining. In some tubules there will be abundant spermatogonial cells, but in others very few.

Primary spermatocytes are a little tricky to identify from spermatogonia when they are first formed. Because they in fact were spermatogonia until they moved into the adluminal compartment. Early primary spermatocytes can have nuclei just like those of spermatogonia in terms of size and density; additionally, the position of their nuclei is just above the basal compartment. You are not expected to be able to identify such early primary spermatocytes!

Soon the primary spermatocytes enlarge in both cytoplasmic and nuclear volume. The cells remain as primary spermatocytes for a prolonged period (about 14 days), during which there are extensive nuclear and cytoplasmic changes. Even the boundaries of the cells differ in how apparent they are at various stages. The cytoplasm stains deeply at times with eosin, but near meiosis becomes very pale and virtually unstained. Experts can distinguish the leptotene, zygotene, pachytene, diplotene and diakenesis subdivisions of nuclear changes during prophase I, but you don't need to consider this problem. The major clue for you to know is that the primary spermatocytes (except quite early ones) have significantly larger nuclei that either spermatogonia or spermatids. The only other large nuclei within the tubules belong to Sertoli cells and these have a different morphology and lie at a different level (and in fact are significantly smaller).

Secondary spermatocytes exist as a very brief stage (a few hours out of the 48 day cycle) and therefore are very rare in slides despite the fact that they are numerically twice as abundant as the easily located primary spermatocytes. These cells are in fact the rarest cell type and consequently, we don't expect you to identify this cell type!

Spermatids and stages of spermiogenesis are the most abundant cell type in testis sections because the cells are numerically abundant, but more importantly because the transformation of spermatids to spermatozoa takes about 19 days. Initially spermatid nuclei are quite round; their abundance, small size (about 2/3rds that of the nuclei of primary oocytes or less), pale staining properties (they are haploid), and position above the level of primary spermatocytes are all important clues. The nuclei don't remain round for long though, progressively elongating and then becoming hook-shaped; their DNA soon undergoes a progressive condensation that is first indicated by the appearance of a dark band of chromatin against one half of the nuclear envelope. The nucleus will progressively decrease in size due to the elimination of nuclear sap, and increase in staining intensity as the DNA becomes more and more condensed. In early spermatids, the golgi apparatus (or its product the acrosomal rudiment) appears as an intense red granule (sometimes with two parts) near the nucleus. The flagellum appears relatively early and in later stages the midpiece is represented as a pink-stained thickening along about the first half of its length. During the later two-thirds of spermiogenesis, irregular blebs of cytoplasm are sloughed off the tail of the differentiating sperm. Much of these wastes are phagocytized by the Sertoli cells.

Fully differentiated spermatozoa may be seen in the lumen of some tubules, but "nearly complete" ones deeply embedded in Sertoli cells may be easier to study. You should be able to identify the nucleus, midpiece, and principal piece, but not much else.

At the end of the exercise, YOU SHOULD:

a. understand the organization of the testis

b. be able to recognize typical examples of the following cell types, based largely on the structure and position of their nuclei:
1. connective tissue cell 5. spermatogonia
2. blood cell (no nuclei!) 6. primary spermatocyte
3. interstitial cells 7. various spermatids
4. myoid cells 8. mature sperm 

c. You should also know which cells are haploid and which cells are diploid.

Grasshopper Testis

Examine a slide of grasshopper testis. Try to find an area in the section that shows a testicular lobe cut in longitudinal section. Can you apply what you know about the organization of rat testis to understand the organization of grasshopper testis? Can you identify any cell types in the testis (remember position, cell size, and the number of chromosomes in the cell) should all help you identify some cell types. .

Fetal Testis

Now that you have some understanding of the organization of the mature testis, we will examine a section of human fetal testis. Prior to sexual maturity, the testis has seminiferous cords rather than seminiferous tubules. Examine a thin section of HUMAN FETAL TESTIS under low power. Along two sides of the testis is a thick layer of tissue containing sections of well defined ducts (parts of the epididymis) and some large blood vessels. At the surface of the testis is a well defined "germinal" epithelium and the tunica albuginea. The interior is about equally divided between a) a background meshwork of cells derived from intermediate mesoderm and b) an array of seminiferous cords which are embedded within this meshwork. The meshwork is slightly darker staining and its cells will eventually be differentiated as connective tissue, blood vessels, myoid cells, and interstitial cells. Examination of the cords under higher power reveals that they are essentially a disorganized jumble of cells. You should be able to recognize two types of nuclei within the cords. The first type is rather uniformly speckled and probably the most abundant. These nuclei belong to cells that will become Sertoli cells. The second type of nucleus is larger containing a few scattered clumps of chromatin and often a conspicuous red nucleolus. This second type of nucleus belongs to early spermatogonia derived from primordial germ cells.

Spermatozoa

Examine the slides of Frog, Human, and Chicken sperm. Identify the head, midpiece, and tail of each sperm. How does their morphology compare to the spermatozoa in the rat and grasshopper testes? How does the spermatozoa of the Crayfish testis compare to all the other sperm cells you have seen?

OVARIES AND OOGENESIS

We will begin our studies of ovaries and oogenesis by examining section of monkey ovary. Obtain one of the thin (1.5 micrometer) sections of monkey ovary labeled OVARY, MONKEY or MONKEY OVARIAN FOLLICLES. Begin your study using the low power objective and note that the ovary is roughly oval in shape with a thin sheet of tissue attached at one end. The latter is the mesovarium, the mesentery that attached the ovary to the body wall and is the route for blood vessels to and from the ovary. The ovary itself is divided into a central medulla and a more peripheral cortex. The medulla consists largely of connective tissue and blood vessels and appears more or less continuous with the mesovarium. The cortex is the "business" part of the ovary and will be the focus of our attention.

Begin your study of the cortex at the ovarian surface. A sheet of squamous (flattened) to cuboidal cells, inappropriately called the "germinal" epithelium, forms the outer surface. The germinal epithelium is underlain by a layer of connective tissue, that some authorities call the tunica albuginea. Other authorities claim the true tunica albuginea (homologous with that bounding the testis) lies between the cortical and medullary layers. Internal to this tunic are numerous early follicles, each surrounded by a single layer of squamous follicle cells and embedded in the "stroma" of the cortex. The term stroma is used for filling or parenchymal cells of the ovary, as well as of other organs including the uterus and testis. Stromal cells of the ovary are essentially unspecialized intermediate mesoderm cells. Some of the stromal cells become modified as thecal cells around each follicle and others are modified as interstitial cells. Interstitial cells are abundant in some organisms and apparently produce hormones (as do the interstitial cells of the testis).

Examine the cortical region of the ovary near the germinal epithelium. Look for large cells with a big nucleus. Most of the follicles are resting or primordial follicles characterized by the enveloping single layer of flattened or squamous follicle cells.  Examine the peripherial region of cortex follicular cells. Under high power examine a good example of such a primordial follicle, selecting one in which the nucleus and its single large nucleolus are included in the plane of section. The nucleus is somewhat excentric and enlarged as is characteristic of primary oocytes. The germinal vesicle (nucleus) does not stain intensely blue because the chromatin (DNA) is so dispersed within the abundant nuclear sap. Near the nucleus is a group of small pink granules; in electron micrographs these cytoplasmic granules are seen to include a large Golgi body and numerous mitochondria.

Some of the follicles have become active, growing early primary follicles that can be distinguished because they have cuboidal to columnar follicle cells rather than flattened ones. The enlarged follicle cells are initially in a single layer, but will soon proliferate mitotically to form multiple layers (middle primary follicle); the follicle cells are called granulosa cells after proliferation begins. While in the primary or preantral phase, follicles increase noticeably in size and complexity. In a late primary follicle, a) the ovum has increased in size and is now separated from the granulosa cells by the zona pellucida; b) the granulosa cells are several layers thick and are delimited from the surrounding cells by the noncellular membrane propria (this is not obvious in our preparations due to the staining used); and c) the stromal cells adjacent to the follicle form a specialized sheath or theca around it. (You should be aware that in the preeding description "primary follicle" is used for all growing preantral follicles while many other descriptions use a more complicated terminology.)

The primary or preantral follicles are converted to antral follicles when the follicle cells secrete follicular fluid; this liquid collects in small pools initially, but these soon fuse as a single antral cavity or antrum. This cavity is crescentic in section since the oocyte and a surrounding "cloud" of granulosa cells are located on one side of the follicular cavity. The oocyte, zona pellucida, and surrounding granulosa cells collectively are called the cumulus oophorus. Even though each of the available sections have about a half dozen antral follicles, usually only one of these has any part of the oocyte and in only a few slides is the oocyte nucleus sectioned (can you explain why this is true?).

Examine the theca of a large antral follicle and note that the more superficial cells are spindle-shaped and arranged as a more or less discrete capsule the theca externa. The thecal cells between the theca externa and the granulosa cells are collectively called the theca interna; the theca interna is richly vascularized (note the capillaries with blood cells) and its cells are secretory. From lecture recall what these cells secrete.

Note that the antral follicles are situated relatively deeply within the cortex. In the final stage of development, the mature or Graafian follicle reaches its full size and then will extend more than the full width of the cortex, pushing into the medulla and bulging out the surface of the ovary; examine a slide of a Graafian follicle. Ovulation will involve the rupture of the ovarian surface as well as the wall of the follicle (theca interna, theca externa, membrana propria and the layer of granulosa cells. Recell that in humans, the first meiotic division occurs shortly before ovulation. The secondary oocyte is released at metaphase of the second meiotic division; it and the first polar body are still contained within the zona pellucida. The latter is surrounded by a halo of granulosa cells which are collectively called the zona radiate. (In some texts the adherent granulosa cells are said to comprise both the zona radiate and a cumulus oophorus layer.) This composite group of cells and the follicular fluid are released at ovulation.

Atretic follicles

Only a fraction of the oocytes which begin development are successful in being ovulated. The rest are aborted somewhere during their development, most frequently in the preantral phase. Examine under low power a slide labeled OVARIAN FOLLICLES, MONKEY. Atresia of follicles begins with degeneration of the oocyte itself. In our slides this process has been completed, but the zona pellucida is still present, although it is collapsed upon itself since the oocyte is no longer there. Find an atretic follicle containing an empty and collapsed zona pellucida. The atretic follicles in our slides have also suffered resorbtion of most of the granulosa cells, but the theca interna cells have enlarged and are now filled with lipid droplets, giving them a pale staining appearance. These "luteinized" thecal cells are arranged in radial cords or strands, partly separated from each other by connective tissue and capillaries. Eventually the atretic follicle will be resorbed, leaving only a small scar within the ovary.

The Fetal Ovary

Examine a slide of the ovary of a human fetus (OVARY, HUMAN FETUS) to demonstrate the great differences between the developing and functional ovary. Try to identify germ cells and somatic cells.

Frog and Chicken Ovaries

Compare the structure of mammalian ovaries of to those of frogs and chickens. Do you see similar structures? Examine a primary oocyte in a frog ovary. Look carefully at the nucleus. Do you see some darkly staining structures? These structures are nucleoli. What is a nucleolus and what happens there?

Frog Ovary Frog Ovary

Examine a section of chicken (hen) ovary. Try to identify the earliest primary oocyte. What do you think the size difference is between this cell and that of a fully developed primary oocyte?

Grasshopper and Honey Bee Ovaries

One problem that organisms have to solve is how to provide eggs with yolk and other nutrients to support development. Grasshoppers and honey bees have solved this problem differently. Honey bees have specialized accessory cells connected to the oocyte called nurse cells that help to pack nutrients into eggs. Grasshoppers do not have nurse cells associated with their oocytes. Examine your slide of grasshopper ovary and determine the organization of primary oocytes and their follicle cells. Next, examine the slide of honey bee ovary that is on demonstration and determine the organization of primary oocytes, nurse cells, and follicle cells.

Egg Maturation: Meisois in Ascaris

Ascaris is a nematode - what kind of organism is that? In Ascaris, sperm-egg fusion occurs while primary oocytes are in the uterus. Consequently, the events of egg maturation (meiosis) overlap the events of fertilization. Examine a section of Ascaris uterus and identify oocytes that are at first meiotic metaphase and second meiotic metaphase.