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Chapter 11: Dynamics of ovarian function: folliculogenesis, oogenesis, and ovulation.

CHAPTER OBJECTIVES

* Describe phases of follicle development.

* Describe mechanisms regulating follicle development.

* Describe synthesis of estradiol by follicles.

* Describe development of the oocyte.

* Describe the mechanism of ovulation.

OVERVIEW

The ovarian cycle facilitates fertilization of an oocyte via four important processes:

* development of an oocyte that can be fertilized (oogenesis),

* development of a preovulatory follicle that will respond to an LH surge (folliculogenesis),

* release of the oocyte contained within the follicle (ovulation), and

* preparation of the reproductive tract for transport of gametes.

Although these processes can be studied separately, they are intimately related. For example, follicular cells support oogenesis, whereas ovulation requires development of a mature follicle. In addition, the hormones produced by follicular cells influence the motility of the reproductive tract which is important in gamete transport. In this chapter we will explore the mechanisms whereby follicles and oocytes develop and ovulate.

EARLY DEVELOPMENT OF THE OOCYTE AND FOLLICLE

Oogenesis and folliculogenesis are intimately associated (Figure 11-1). Each of these processes begins during sexual differentiation, when primordial germ cells infiltrate the embryonic gonad. The final stages of oogenesis and folliculogenesis occur in the adult during the follicular phase of the ovarian cycle. Oogenesis begins in the fetal ovary when the primordial germ cells enter the gonad and become oogonia. These cells proliferate via mitosis during fetal development. Toward the end of pregnancy, all of the oogonia begin meiosis and become primary oocytes. Further development of these cells is arrested during the first meiotic prophase. Thus at the time of birth the ovaries contain all the oocytes they will ever have. In most mammalian species, completion of the first part of meiosis occurs at ovulation, whereas the second meiotic division is completed upon fertilization. Unlike spermatogenesis, oogenesis in the adult does not increase the number of gametes. The first and second meiotic divisions produce the first and second polar bodies, which are small cells that are not viable and eventually regress.

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Follicle development begins about the time oogonia enter the first meiotic prophase. During the early stages of meiosis, the primary oocyte becomes surrounded by a single layer of follicular cells. These cells produce a thin basement membrane, which surrounds the oocyte. At this point the oocyte, along with the follicular cells and basement membrane, is known as a primordial follicle. It is believed that follicular cells generate a meiosis inhibitory factor, which keeps the oocyte in an arrested state of development until ovulation. Development of the oocyte ceases soon after the follicular cells condense around the oocyte. Gap junctions form between the oocyte and the follicular cells that surround it. Inhibition of oocyte maturation depends on a cell-to-cell communication facilitated by these gap junctions. Resumption of oocyte development occurs near the time of ovulation and is related to disruption of gap junctions brought about by the preovulatory surge of LH.

Primordial follicles can remain in this state of suspended animation for many years. For example, human females do not begin ovulating until the end of the first decade of life and continue expressing ovarian cycles until 50 years of age or more.

FOLLICULOGENESIS

A primordial follicle has three possible fates. First, it can remain quiescent. Some primordial follicles never resume development. Second, the follicle can undergo atresia. This is what happens to the vast majority of follicles. Third, the follicle can resume development. There are two possible fates for follicles that resume development. Most undergo atresia. A few develop to the fully mature state and ovulate.

The amount of time required for a follicle to grow to a preovulatory size is much longer than a single ovarian cycle. For example, in humans (Figure 11-2) this process takes almost 1 year. Follicle growth is typically divided into three major phases: preantral, antral, and preovulatory. The preantral phase refers to the period of development when the follicle lacks an antrum (primordial, primary, and secondary follicles). This phase begins when a primordial follicle enters a pool of developing follicles, becomes a primary follicle, and then develops multiple layers of granulosa cells to become a secondary follicle. The preantral phase is the longest of the three phases, but less is known about this stage than the others. The antral phase of follicular growth begins when a secondary follicle begins to develop a fluid-filled antrum to become a tertiary follicle. During this phase of development, follicular cells proliferate and produce increasing amounts of follicular fluid, which causes the size of the follicle to expand. The end of this phase is not as clearly demarcated as the transition between the preantral and antral phases and depends on the prevailing hormonal conditions. For most tertiary follicles, the end of the antral phase is marked by atresia. The few tertiary follicles that do not become atretic enter the preovulatory phase. This phase is known as selection. During this final phase, one follicle experiences rapid growth and becomes the ovulatory follicle (maturation).

[FIGURE 11-2 OMITTED]

Preantral Phase

As noted earlier, the preantral phase of folliculogenesis begins with the growth of primordial follicles. During this phase, follicular diameter increases from 20 to 400 [micro]m due largely to growth of the primary oocyte. Although the oocyte is very active during this phase, it remains in the first meiotic prophase. Some of the more significant activities include RNA synthesis, development of a dense nucleolus in the nucleus, and loading of the cytoplasm with organelles (Golgi apparatus and endoplasmic reticulum). Such changes are prerequisites for later stages of oocyte development that occur following ovulation.

Changes in follicular cells also contribute to follicle growth during the preantral phase. The granulosa cells surrounding the oocyte divide and form multiple layers. The cells that are in contact with the oocyte become the corona radiata and secrete a glycoprotein that becomes part of the zona pellucida, a membrane that surrounds the oocyte. Cytoplasmic processes project from the innermost layer of granulosa cells through the zona pellucida and form gap junctions with the oocyte membrane. This creates a system through which the granulosa cells provide various low-molecular weight substrates (e.g., amino acids and nucleotides) to the oocyte. These compounds are the building blocks for important macromolecules such as proteins and nucleic acids. A basement membrane develops and encases the outermost layer of granulosa cells. Loosely organized layers of spindle-shaped cells develop around the layer of granulosa cells and become known as thecal cells. Unlike the granulosa cell layer, the layer of thecal cells becomes richly supplied with capillaries.

Antral Phase

Resumption of follicle development occurs sporadically and incompletely during prenatal and neonatal life. During these periods a few follicles may develop to the antral stage, but they soon regress. As females approach puberty, initiation of follicle growth becomes more common. In the adult female, primordial follicles enter a pool of growing follicles at a steady trickle. At regular intervals, a group of follicles enters the antral stage and these follicles grow in wave-like patterns. Briefly, a follicular wave is the pattern of growth expressed by a follicle during the antral phase of development. Typically the diameter of an antral follicle increases until it ovulates or undergoes atresia. The number of follicle waves varies among and within species. In primates, growth of antral follicles is restricted to the follicular phase of the menstrual cycle. In contrast the cow, the ewe and the mare can express more than one follicular wave during a particular estrous cycle (Figure 11-3). For example, two- or three-wave estrous cycles predominate in various herds of cattle. In cows, a follicular wave consists of the emergence of a group of antral follicles, approximately 4 mm in diameter, followed by growth of all follicles and then continued growth of only the largest follicle. The wave that ends with ovulation is typically referred to as the ovulatory wave, whereas waves that do not lead to ovulation are known as anovulatory waves.

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Selection of the Ovulatory Follicle

One of the most fundamentally important questions associated with folliculogenesis is, how is the preovulatory follicle selected from the vast pool of follicles? Figure 11-4 summarizes the events leading to selection of the ovulatory follicle in humans. It appears that the same sequence of events applies to other species as well.

Events leading to the selection of the preovulatory follicle begin during the preantral phase of follicular development. At any given time, there is a group of secondary follicles that have completed preantral growth and developed receptors for LH and FSH. If concentrations of these gonadotropins are sufficient, secondary follicles will develop into antral follicles. This process is known as emergence or recruitment. Recruitment of a group of follicles prevents additional follicles from entering the pool of antral follicles.

The fate of the future dominant follicle is sealed during emergence. Even though antral follicles are at similar stages of development at the time of emergence, there is variation in their rates of development. The follicle developing at the fastest rate will be the one that ultimately becomes the dominant follicle and ovulates. In cattle, this future dominant follicle emerges from the pool of secondary follicles at least 6 to 7 hours before the other follicles when it is only 3 to 4 mm in diameter. This phase of development is commonly referred to as selection; that is, when the largest follicle emerges from the pool of tertiary follicles. Antral follicles grow in parallel such that the largest follicle maintains a slightly larger diameter than other follicles. In cattle, the diameter of the future dominant follicle remains approximately 0.5 mm larger than that of the next largest follicle.

During the antral phase, both the granulosa and theca cells proliferate, but there is little if any change in the size of the oocyte. The thecal cells form two distinct layers: a richly vascularized inner layer of endocrine cells (theca interna) separated from a connective tissue layer (theca externa) by a fibrous capsule. As the granulosa cells proliferate they produce follicular fluid, which accumulates between the cells and eventually displaces them, forming a fluid-filled antrum. A layer of granulosa cells known as the cumulus oophorus surrounds the oocyte, which is suspended by a thin column of granulosa cells.

Preovulatory Phase

The end of the antral phase of folliculogenesis is marked by an event known as deviation (Figure 11-4). During deviation, the largest follicle continues to grow and smaller follicles experience a reduction or cessation of growth. It is at this stage when a dominant follicle can be clearly identified. In cattle, deviation occurs within 2.5 days after emergence of the future dominant follicle. Growth of the dominant follicle is rapid during the preovulatory phase. The tremendous increase in follicle diameter is due primarily to expansion of the volume of follicular fluid. In addition, production of estradiol by follicular cells is reaching a maximum level during this time. The preovulatory phase ends with ovulation.

Regulation of Folliculogenesis

Follicle growth can be divided into two phases based on how the process is regulated; that is, gonadotropin-independent and gonadotropin-dependent (Figure 11-5). Preantral follicle growth is not dependent on gonadotropins, whereas LH and FSH direct follicle growth during the antral and preovulatory phases.

Gonadotropin-Independent Follicle Growth

The preantral phase of follicle growth is not disrupted by hypophysectomy (removal of the pituitary gland). Thus it appears that development of primary and secondary follicles does not depend on gonadotropins. Although the mechanisms responsible for development of follicles during the preantral phase are not well understood, it is clear that this phase of development is orchestrated by a variety of hormones produced in the ovary and acting via paracrine, neurocrine, and autocrine mechanisms. It is noteworthy that receptors for LH and FSH do not appear on follicular cells until the end of the preantral phase; that is, on secondary follicles. In fact, the appearance of these receptors is a prerequisite for emergence and subsequent follicle growth.

[FIGURE 11-5 OMITTED]

Gonadotropin-Dependent Follicle Growth

Follicle growth during the antral and preovulatory phases is regulated largely by the actions of FSH and LH. In general, FSH plays a pivotal role in follicle development between emergence and deviation, whereas LH is most important during postdeviation growth (Figures 11-5 and 11-6).

ROLE OF FSH An increase in FSH is required to induce recruitment of follicles (Figure 11-6). A surge of FSH precedes and initiates the occurrence of a follicular wave in cows, horses, and sheep. In primates, the gradual increase in FSH concentrations observed during the early follicular phase is sufficient to induce recruitment of antral follicles. The growth of all recruited follicles continues as concentrations of FSH decline following the surge and this growth appears to require FSH.

Concentrations of FSH decline for several days following the peak of the FSH surge. This decrease in FSH continues through selection and deviation and appears to play a critical role in regulating these processes. This is supported by the observation that injections of FSH prevent or delay deviation in cows.

[FIGURE 11-6 OMITTED]

The mechanism causing the decrease in FSH concentrations following the peak in FSH are complex. Initially, the decrease may be due to depletion of releasable pools of FSH in pituitary gonadotropes. In addition, negative feedback signals generated by growing follicles play a major role in regulating this response. In cows, antral follicles develop an FSH-depressing ability within 1 day after emergence and contribute to the drop in FSH for about 2 days. Between 2 and 3 days after emergence the largest follicle develops an enhanced capacity to suppress FSH. As a result the concentration of this gonadotropin falls below that required to sustain the growth of smaller follicles. Apparently, the largest follicle is more sensitive to FSH than the smaller follicles and continues to develop even in the presence of lower FSH concentrations. The negative feedback signal mediating the inhibitory effect of the follicles on FSH release appears to include both estradiol and inhibin. Follicular production of these hormones increases greatly as follicles develop. The enhanced sensitivity of the largest follicle to FSH may be attributed to its larger surface area, and thus a greater number of FSH receptors, and/or intrafollicular factors (e.g., estradiol), which enhance responsiveness to FSH. In summary, the largest follicle induces deviation by suppressing concentrations of FSH while retaining the ability to respond to decreasing concentrations of FSH. The fact that the largest follicle grows while suppressing development of smaller follicles serves as the basis for calling the largest follicle the dominant follicle.

ROLE OF LH LH plays an important role in maturation of the dominant follicle following deviation (Figures 11-6 and 11-7). Continued growth of this follicle following deviation is an LH-dependent process. This idea is supported by the fact that suppression of LH following deviation stalls follicular growth, thereby preventing follicles from reaching a preovulatory size. In cows, the effects of LH are mediated by the appearance of LH receptors on the granulosa cells of the largest follicle between 2 and 4 days after emergence, the time when deviation begins. Before this stage, LH receptors are found only in the thecal cells (Figure 11-7). The appearance of LH receptors in granulosa cells permits these cells to respond to both LH and FSH (Figure 11-7). In light of the fact that FSH concentrations decrease during deviation, it appears that LH becomes an important regulator of follicular growth during this period. It is likely that the combined effects of LH and FSH account for the rapid growth and enhanced activity of the dominant follicle. As the largest follicle attains dominance, production of estradiol increases dramatically. The enhanced production of estradiol by the dominant follicle and the appearance of LH receptors on its granulosa cells are necessary conditions for ovulation. However, these events are not sufficient conditions for ovulation. Ovulation also requires the estradiol-induced LH surge.

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The fate of the dominant follicle depends on the prevailing hormonal environment. If the dominant follicle develops at a time when there is no corpus luteum and progesterone concentrations are low, then the elevated concentrations of estradiol induce an LH surge, which causes the dominant follicle to ovulate. Follicle waves that reach a crest in the presence of a corpus luteum will not ovulate due to the fact that high levels of progesterone block the positive feedback effects of estradiol on LH. Thus neither the LH surge nor ovulation can occur in the presence of a corpus luteum. In these cases, the dominant follicle undergoes atresia and regresses.
BOX 11-1 Focus on Fertility: Superovulation and Embryo Transfer

Embryo transfer was the next assisted reproduction
technology developed after artificial insemination.
Although the use of this technology is not as
widespread as and often less successful than
artificial insemination it is employed routinely in
several species of livestock, species of wildlife,
and humans. Embryo transfer can be used to
accelerate proliferation of genetic material from
females, minimize spread of reproductively transmitted
diseases, salvage genetic material of valuable
individuals, and facilitate development of new
lines or breeds of livestock. The general paradigm
for successful embryo transfer in cattle is shown in
Figure 11-8. Today embryos are routinely recovered
nonsurgically by flushing them from the uterine
horns several days after fertilization (when they
are at the blastocyst stage of development). They
can be recovered and immediately transferred to
recipients, but this requires that the recipients be
at the same stage of the ovarian cycle as the
donors (7 days after estrus). It is also possible to
subject embryos to cryopreservation and store
them for transfer at later times. Embryos can also
be prepared by in vitro fertilization, which requires
mixing of spermatozoa and an oocyte in a Petri
dish and allowing the embryo develop to the
blastocyst stage before transfer.

[FIGURE 11-8 OMITTED]

The success of embryo transfer is heavily
dependent on the ability to produce and recover
viable embryos from donor females. In order to
enhance the possibility of achieving this goal, donors
are typically subjected to hormone treatments
that superstimulate the ovaries to induce multiple
ovulations (Figure 11-9). This technique is called
superovulation. The basic concept underlying this
method is that the number of follicles that
undergo deviation and become dominant can be
increased by boosting the number of follicles that
are recruited. Enhancement of recruitment is
accomplished by administering FSH during the late
luteal phase, the time when follicles are recruited
for the next cycle. In most cases, donor cows are
treated with [PGF.sub.2[alpha]] toward the end of FSH
treatments in order to induce estrus and ovulation
at predictable times. This facilitates use of
artificial insemination. Response to super-ovulation
regimens are quite variable producing anywhere
between 0 and 20 (or more) ovulations. The
average response is nine ovulations.

[FIGURE 11-9 OMITTED]


ENDOCRINE ACTIVITY OF FOLLICLES

In addition to regulating folliculogenesis and oogenesis, the gonadotropins interact to regulate the endocrine activity of follicles (see Figure 11-7). During follicle selection, LH acts on theca interna cells to promote conversion of cholesterol to testosterone. Recall that this effect is similar to that in the Leydig cells of the testes. Testosterone is released into the extracellular fluid and can be taken up into the blood to enter the general circulation, or diffuse across the follicular wall and be taken up by granulosa cells. In the granulosa cells, testosterone is the substrate for the aromatase enzyme, which converts it to estradiol. This enzyme is regulated by FSH. Estradiol is released by the granulosa cells into the follicular fluid. A small portion of this hormone can diffuse out of the follicle and enter the blood.

The regulation of steroid hormone production by the follicle changes during deviation. Recall that the dominant follicle is the first one to develop LH receptors on granulosa cells. At this point the granulosa cells are stimulated by the combined effects of LH and FSH. This causes rapid growth of the follicle and elevated secretion of estradiol and inhibin. Note that the effects of LH and FSH on estradiol synthesis are additive due to the fact that both hormones act by increasing synthesis of cAMP. It is also important to point out that the increased production of estradiol and inhibin by the dominant follicle exert negative feedback effects on FSH release. The resulting decrease in FSH prevents recruitment of new follicles and is ultimately responsible for the slowed growth and atresia of the nondominant follicles. The decrease in FSH has no inhibitory effect on the dominant follicle due to the ability of its granulosa cells to respond to LH.

OOGENESIS

As noted earlier, development of the oocyte begins before birth, but then ceases until the time of ovulation. Figure 11-1 summarizes the major aspects of oogenesis as related to follicle development, ovulation, and fertilization. Figure 11-10 summarizes highlights of oogenesis with reference to mitosis and meiosis. Once the primordial germ cells enter the gonad of female embryos, they differentiate into oogonia. These cells undergo a series of mitotic divisions before entering meiosis. By the time of birth the oogonia enter the first meiotic prophase and are then referred to as primary oocytes. This phase of meiosis is completed only to the diplotene stage. Although the primary oocyte enlarges greatly at this point, it exists in this arrested state (resting phase) of development until ovulation. An important implication of this phenomenon is that female mammals achieve a maximum number of oocytes at some point during the fetal stage of development (a peak number of 7 million by midge-station in humans). Thereafter, the number of oocytes decreases progressively due to the loss of follicles by atresia. The growth of the primary oocyte is passive; that is, not dependent on follicular cells. As the oocyte grows it becomes enveloped by the zona pellucida, a translucent, jelly-like membrane consisting of mucopolysaccharides and proteins. This membrane forms once the oocyte is surrounded by a layer of cuboidal granulosa cells. The zona pellucida is not solid. Rather it contains microscopic canals through which microvilli from the adjacent granulosa cells extend and terminate in indentations of the oocyte. This structural arrangement seems to provide a means for transport of metabolites from granulosa cells to the oocyte and persists until ovulation.

[FIGURE 11-10 OMITTED]

[FIGURE 11-11 OMITTED]

Up until the point of ovulation, development of the oocyte is gonadotropin-independent. The first meiotic division is completed at ovulation and appears to be induced by the LH surge. Completion of this phase results in formation of a secondary oocyte and a first polar body. The polar body is a small offspring cell that has substantially less cytoplasm than the secondary oocyte. It eventually degenerates.

The mechanism whereby the LH surge liberates the primary oocyte from meiotic arrest is quite elegant and involves structural changes in the relationship between the oocyte and cells of the cumulus oophorus that send cytoplasmic projections through the zona pellucida (Figure 11-11). Before the LH surge, gap junctions between the cumulus cells and the oocyte permit direct chemical communication between the cells. The cumulus cells produce oocyte maturation inhibitor (OMI), which enters the cytoplasm of the oocyte and inhibits activation of another protein known as maturation promoting factor (MPF). The LH surge destroys the gap junctions between the cumulus cells and oocyte, thus lowering concentrations of OMI. The decrease in OMI allows MPF to be produced and this protein acts on the nucleus of the oocyte to induce completion of the first meiotic division.

Immediately after completion of the first meiotic division, the secondary oocyte enters the second meiotic prophase. Completion of meiosis depends on fertilization by a spermatozoon. The second meiotic division results in formation of an ootid and a second polar body.

OVULATION

Not only is ovulation a pivotal event in the ovarian cycle, it is arguably the most important rate-limiting process in the overall reproductive fitness of a species. As noted in previous discussions, ovulation serves as a convenient point of demarcation that separates the cycle into two major portions: follicular and luteal phases. The rupture of the dominant, preovulatory follicle and release of its oocyte is also a critical physiologic event that is necessary for the female gamete to become available for fertilization. Two functionally independent events occur in association with ovulation: rupture of the follicular wall and luteinization. The former is concerned with release of the oocyte from the ovulatory follicle, whereas the latter is concerned with transformation of follicular cells into cells of the corpus luteum. Both of these processes are triggered by the pre-ovulatory surge of LH.

During the past 75 years several hypotheses have been put forth to explain how the LH surge brings about rupture of the ovulatory follicle. By the early 1960s, it was generally assumed that ovulation was the result of increasing intrafollicular pressure brought about by contraction of the smooth muscle cells located in the ovarian stroma. This hypothesis is no longer tenable. There is little evidence to support the idea that smooth muscle cells exist in the connective tissue surrounding follicles, let alone that these cells contract during ovulation. Moreover, pressure within the follicle does not increase before ovulation. Ovulation appears to be the result of a weakening of the follicular wall under the force of modest but steady intrafollicular pressure, which is primarily due to the hydrostatic pressure of capillaries in the theca interna.

Anatomic Changes in the Follicle Before and During Ovulation

An understanding of the mechanism of ovulation requires familiarity with the histology of the follicular wall (Figure 11-12). The selective growth of the pre-ovulatory follicle causes the outer tissue layer of the follicule to come into close apposition with the surface epithelium and underlying capsule (tunica albuginea) of the ovary. The site is known as the apex of the follicle, and marks the location of ovulation. When the follicle wall in this region begins to weaken, a stigma forms and designates the site at which the follicular wall will rupture.

The apical follicular wall has a complex structure consisting of five tissue layers. As we study the ovulatory process, keep in mind that each of these layers must be breached in order for the oocyte to be released from the follicle. The first, or outermost layer, is the single layer of cuboidal epithelial cells known as the germinal epithelium. The second layer is the tunica albuginea, a connective tissue sheath consisting of fibroblasts and collagen fibers. This layer is between 5 and 7 cells deep and envelopes the entire ovary to delineate its integrity. The theca externa forms the third layer of the follicle wall. This forms the follicle's own layer of connective tissue and includes several layers of fibroblasts and collagen fibers. It may be difficult to distinguish a border between the tunica albuginea and theca externa due to the fact that the two layers mesh together. The fourth layer is the theca interna. This layer consists of two layers of elongated cells that contain numerous mitochondria, lipid droplets, and a well-developed smooth endoplasmic reticulum, which are organelles involved in production of steroid hormones. The theca interna contains most of the capillaries supplying blood to the follicle. The fifth, and innermost tissue layer consists of granulosa cells. These cells are attached to a basal lamina which separates the theca interna from the granulosa layer. The granulosa layer is avascular because the capillaries of the theca interna do not penetrate the basement membrane. On average, the granulosa layer is five to seven cells thick. However, additional layers of cells exist at the cumulus oophorus, a pedestal of granulosa cells that support the oocyte. Gap junctions between adjacent granulosa cells create a functional syncytium. Gap junctions also exist between the oocyte and the cells of the corona radiata. This organization of cells coordinates activity of granulosa cells and permits direct communication between and oocyte and this layer of follicular cells.

[FIGURE 11-12 OMITTED]

The Ovulatory Process

Complete rupture of the ovulatory follicle and release of the oocyte requires between 10 and 40 hours, depending on the species. Changes in the ultrastructure of the follicular wall can be observed during the final hour before ovulation. The most important of these changes include the following:

* Fibroblasts of the tunica albuginea and theca externa change from a quiescent, resting to an active, proliferating state, and begin to dissociate from one another.

* Connective tissue of the tunica albuginea and theca externa at the apex of the follicle becomes more loosely organized and less tenacious.

* Cuboidal cells of the germinal epithelium develop vacuoles and become necrotic.

* Theca interna cells remain unchanged but some of the capillaries contain coagulated blood causing petechia on the surface of the follicle.

* Granulosa cells accumulate lipid in lipid droplets, reflecting increased synthesis of progesterone.

Additional changes occur within the apex of the follicle a few minutes before ovulation. The most noticeable is that this region of the follicle wall bulges outward, forming the stigma. This event is associated with the following changes in ultrastructure:

* Epithelial cells on the surface of the follicle slough off.

* Cells of the theca interna and granulose layers dissociate and migrate to the base of the stigma.

* The thin layer of highly degraded collagenous tissue that remains at the apex narrows to less than 20 percent of its original thickness.

Ovulation itself requires hydrostatic pressure within the antrum of the follicle. The pressure is modest and does not change throughout the ovulatory process. Rupture of the follicle is attributed to this sustained pressure and the weakening of the follicle wall. Intrafollicular pressure is the hydrostatic pressure of capillaries located within the theca interna. This pressure drops once the follicle ruptures.

Regulation of the Ovulatory Process

It is generally agreed that the major prerequisite for ovulation is degradation of collagenous layers of the theca externa and tunica albuginea at the apex of the ovulatory follicle. However, information about the biochemical events responsible for this response is incomplete. Figure 11-13 summarizes aspects of the ovulatory mechanism for which there is general agreement among reproductive biologists.

As noted earlier, events leading to ovulation are initiated by the LH surge. Receptors for this gonadotropin exist in both the thecal and granulose cells as well as the surface epithelium of the ovary. Thus the mechanism whereby LH induces ovulation is likely to involve a direct effect on each of these cell types. It also appears that the fibroblasts of the tunica albuginea, and theca externa, and the endothelial cells of the theca interna are also involved with ovulation, but regulation of these cells is likely to be dependent on signals generated by cells that respond directly to LH. The overall effect of the LH surge is to terminate expression of genes that govern activity of the pre-ovulatory follicle, and to induce expression of genes that regulate ovulation. Effects on the granulosa cells may be most important in regulating ovulation. Two of the most important ovulation-regulating genes are the gene for the progesterone receptor and the cyclooxygenase-2, an enzyme regulating synthesis of prostaglandins. These signaling pathways act independently to induce changes that promote ovulation and luteinization. With respect to ovulation, some of the more important changes include 1) activation and/or synthesis of proteolytic enzymes (collagenase) that degrade the extracellular matrix and 2) synthesis of regulatory factors that act on fibroblasts and endothelial cells to induce an inflammatory-like cascade of events. These latter changes result in increased blood flow, edema, and localized necrosis of tissue.

[FIGURE 11-13 OMITTED]

In addition to inducing degradation of the apical follicular wall, the LH surge induces changes within the cumulus oophorus that are necessary for ovulation to occur. The LH surge acts on cumulus cells to induce production of various proteins that interact to form a biochemical matrix upon which the cumulus cells move during ovulation.

In summary, release of the oocyte from the pre-ovulatory follicle is the result of three major LH surge-induced events: 1) sustained hydrostatic pressure due to enhanced blood flow to the follicle, 2) collagenase-induced digestion of the follicle wall, and 3) separation of the oocyte from the follicular wall.

Luteinization

Within hours after the LH surge, the tissue of the ovulating follicle begins luteinization; that is, a remodeling process that leads to the formation of a corpus luteum. This process involves both morphologic and biochemical changes. Major changes include the distinct concentric structure of the follicle collapses permitting cells of the various layers to intermingle, the thecal and granulosa cells become reprogrammed to express a luteal pattern of genes, the remaining tissue undergoes rapid growth due to development of an extensive capillary network, and proliferation of various cell types.

SUMMARY OF MAJOR CONCEPTS

* Development of the follicle and oocyte occur in parallel, but the two processes are not necessarily interrelated.

* Folliculogenesis consists of a preantral, antral, and ovulatory phases.

* The preantral phase of folliculogenesis is not dependent on gonadotropins, whereas the antral ovulatory phases are gonadotropin-dependent.

* During the antral phase of folliculogenesis, follicle growth is wave-like due to three processes: recruitment, selection, and deviation.

* LH and FSH act on theca interna cells and granulosa cells, respectively, to promote estradiol synthesis.

* Oogenesis consists of major phases including: expansion of the oogonia population by mitotic divisions, a resting phase where primary oocytes remain in meiotic prophase 1, an LH surge-induce completion of the first meiotic division, and fertilization-induced completion of the second meiotic division.

* The LH surge induces ovulation (rupture of the follicle) and luteinization (transformation of the follicle to a corpus luteum).

DISCUSSION

1. One of the major functions of the testis and ovary is the production of gametes. In each case, gamete production involves both mitosis and meiosis. Make lists of similarities and differences between spermatogenesis and oogenesis.

2. Describe what you would expect to happen if you were to induce an LH surge in a cow on day 5 of her estrous cycle (i.e., after the dominant follicle of the first follicular wave has deviated). Explain your answer.

3. Daily injections of FSH given late in the cow's estrous cycle will result in multiple ovulations. Describe the physiologic mechanism responsible for this observation.

4. Explain how the dominant follicle induces a reduction in FSH concentrations. How is it that this reduction in FSH inhibits growth of other tertiary follicles, but does not impair growth of the dominant follicle?

REFERENCES

Farin, P.W., K. Moore, and M. Drost. 2007. Assisted reproductive technologies in cattle. In R.E. Youngquist and W. R. Threlfall, Current Therapy in Large Animal Theriogenology 2. St. Louis: Saunders, pp. 496-508.

Fortune, J.E. 1986. Bovine theca and granulose cells interact to promote androgen production. Biology of Reproduction 35:292-299.

Fortune, J.E. 1994. Ovarian growth and development in mammals. Biology of Reproduction 50:225-232.

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Keith K. Schillo, PhD

Department of Animal and Food Sciences

University of Kentucky

Lexington, Kentucky
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Author:Schillo, Keith K.
Publication:Reproductive Physiology of Mammals, From Farm to Field and Beyond
Geographic Code:1USA
Date:Jan 1, 2009
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