- Email: [email protected]
Female Reproductive Tract
Chapter 19
Female Reproductive Tract The female reproductive tract comprises the ovaries, oviducts, the uterus and the vagina. Although the rat cervix is not comparable to that found in humans, the junction between the uterus and the vagina is sometimes referred to as the cervix.
DEVELOPMENT AND TOPOGRAPHY The first stages of the development of the ovary are identical to those of the testis, and cannot be distinguished histologically; the later stages are very different. The ovary remains within the abdominal cavity, while the testis descends to the scrotum. The oviducts and uterus are formed from the paramesonephric ducts (Mu¨llerian ducts), which develop from the lining of the peritoneal cavity close to the mesonephric ducts (Wolffian ducts), but have nothing to do with the ‘second kidney’ or mesonephros (see Chapter 18: Male Reproductive System). The paramesonephric ducts are attached to the dorsal abdominal wall by a fold of parietal peritoneum, which develops into the broad ligament of the uterus. The ovary is attached to the surface of the anterior end of the broad ligament by a band of connective tissue known as the mesovarium. In humans, ova released from these surfaces enter the peritoneal cavity, but are quickly swept into the funnel-shaped mouths of the oviducts that run in the broad ligament and open into the peritoneal cavity, very close to the ovary. The oviduct is sometimes called the salpinx (Greek for tube), and the part of the broad ligament that enfolds the salpinx is the mesosalpinx. It will probably be obvious that there is a danger of infection making its way via the vagina, uterus and oviducts into the peritoneal cavity. Infection of the oviduct is referred to as salpingitis, and is one of the most common causes of infertility in women. The anatomy of the rat is a little different, as the ovary is surrounded by a capsule of mesovarium, the ovarian bursa, pierced by the oviduct. The open end of the oviduct lies within it and the ova never actually enter the peritoneal cavity. This arrangement is unusual among
mammals, and some authors maintain that the capsule around the rat ovary is not complete and that a slit connects the interior of the capsule with the peritoneal cavity. The developing ovary, like the developing testis, is attached to the gubernaculum, which itself is attached to the developing uterus. The gubernaculum develops into the round ligament of the ovary, which connects the ovary with the tip of the adjacent uterine horn and the round ligament of the uterus, which connects the uterus with the abdominal wall at the internal inguinal ring. In Chapter 18, Male Reproductive System, we describe how the testis, accompanied by a projection of parietal peritoneum (the processus vaginalis), passes through the inguinal canal and into the scrotum. Things are not so very different in the female. The round ligament of the uterus, accompanied for some distance by a female equivalent of the processus vaginalis (human anatomists call this female equivalent the canal of Nuck), passes through the inguinal canal and into the labia majora of the external genitalia. Although this is what happens in female humans, it would seem that diverticuli of the peritoneum (the equivalents of the processus vaginalis) are not always present in female rats (Hunt, 1924). Hebel and Stromberg (1976) refer to the ligament of the ovary that is derived from the gubernaculum as the ‘proper ligament of the ovary’, placed it medial to the oviduct, and described it as blending with the mesosalpinx at the tip of the uterine horn. This ligament contains mainly smooth muscle fibres. They also describe a suspensory ligament that runs from the ovarian hilum to the crus of the diaphragm. This is presumably equivalent to the suspensory ligament of the human ovary (better described in human anatomy as the infundibulopelvic ligament) that forms that part of the broad ligament running from the ovary to the pelvic wall. Der Schoot and Emmen (1996) have provided an interesting discussion of the suspensory ligaments of mammalian gonads. In humans, the ovary descends to the brim of the pelvis, but in the rat, the two horns of the uterus stretch forwards to the ovaries situated on the dorsal aspect of the abdominal wall.
Anatomy and Histology of the Laboratory Rat in Toxicology and Biomedical Research. DOI: https://doi.org/10.1016/B978-0-12-811837-5.00019-8 © 2019 Elsevier Inc. All rights reserved.
219
220 Anatomy and Histology of the Laboratory Rat in Toxicology and Biomedical Research
The Ovary The ovaries of the rat lie caudal to the kidneys. Hebel and Stromberg (1976) placed the right ovary at the level of lumbar vertebrae 4 5 and the left at 5 6, but Rowett (1952) showed them a little further forward, with the left lying alongside the left kidney (Fig. 19.1). In the mature adult, the ovary measures a 5 3 4 3 3 mm3 and weighs about 60 mg. Blood is supplied to the right ovary by a branch of the abdominal aorta, the right ovarian artery and returned via the right ovarian vein to the posterior vena cava. On the left, the vessels are connected with the renal vessels. The ovarian arteries run in the broad ligament and anastomose with the uterine arteries, which arise from the iliac arteries.
Oviduct The oviduct is a narrow looped structure, 0.4 0.5 mm in diameter and 18 30 mm in length, lying adjacent to the ovary, with a funnel-shaped proximal end that pierces the ovarian bursa (Hebel and Stromberg, 1976). The distal end penetrates the tip of the uterus. The mucosa of the proximal part of the oviduct is thrown into a series of branching longitudinal folds, which in cross section appear to almost fill the lumen, extending beyond the open cranial end of the oviduct as finger-like
processes called fimbria. The folds become less prominent as the uterus is approached. The mucosa is a simple columnar epithelium, with both ciliated and unciliated secretory cells. The ciliated cells are said to respond to oestrogen by increasing the frequency of ciliary beating at the time of ovulation. Beneath the epithelium a very vascular lamina propria contains large lymphatic capillaries. There is little evidence of a muscularis mucosae, though some smooth muscle fibres extend into the longitudinal folds. The wall of the oviduct contains a well-developed circular or spiral layer smooth muscle (the muscularis), and a less welldeveloped outer layer of longitudinally disposed smooth muscle. Rhythmic pumping of the tube aids transport of the fertilised ovum to the uterus. It is important to note that fertilisation, the entry of a spermatozoon into the ovum, occurs in the oviduct. The wall of the oviduct is very vascular with a well-developed layer of blood vessels between the circular and longitudinal layers of smooth muscle.
Uterus The rat has a duplex uterus that is it is divided into two separate horns. The horns are 30 40 mm in length and join about 7 10 mm above the vagina, but each horn enters the vagina individually, so functionally the rat can FIGURE 19.1 Dissection to show female reproductive system.
Adrenal gland Fat Kidney Ovary
Ureter
Uterus Mesovarium of oviduct
Bladder Vagina Cut pelvic girdle Vagina Anus Clitoris
Female Reproductive Tract Chapter | 19
be considered to have two uteri. Each opening of a uterine horn into the vagina is surrounded by a small ventral cushion, a large dorsal cushion and two lateral cushions of tissue. Hebel and Stromberg (1976) refer to this as the ‘portio vaginalis uteri’, the doorway from the uterus into the vagina. A short sagittal partition separates the entry points of the horns into the vagina.
Vagina The rat vagina opens to the exterior just in front of the anus and behind the opening of the urethra. The vagina is thin walled, 15 20 mm in length and 3 5 mm in diameter, with a mucosa that is thrown into longitudinal folds.
HISTOLOGY The surface of the ovary is covered by a simple epithelium, once called the germinal epithelium as it was thought that it was the source of the germ cells. This concept has long since been abandoned, and the epithelium is now called the ovarian surface epithelium (OSE). It is derived from the lining of the peritoneal cavity and is really modified parietal peritoneum. The cells of the epithelium vary from squamous to cuboidal, and play an important part in repairing the breach made in the surface of the ovary when ovulation occurs. The OSE has attracted much interest during the past 30 or so years because .80% of human ovarian cancers arise from this epithelium. The OSE has been reviewed in detail by Auersperg et al. (2001) and with regard to the rat by Gayta´n et al. (2005). Beneath the OSE lies the tunica albuginea, a layer of connective tissue containing many fibroblast-like cells. The collagen fibres of the tunica are thin and less well developed than those of the testes. The remainder of the ovary can be divided into a cortex and a medulla. The cortex contains the follicles, the medulla and the large blood vessels running to and from the ovary at the hilum. Between the follicles of the cortex and the blood vessels of the medulla is the ovarian stroma, a fibroblast-rich connective tissue.
Follicles Primordial Follicle Immediately beneath the tunica albuginea lie the primordial follicles. These comprise a primary oocyte surrounded by a single layer of squamous follicular cells. The oocyte is about 25 µm in diameter with a welldefined nucleus, and a nucleolus that often stains pink in H&E stained sections. The development of the primary oocyte arrests during prophase of the first meiotic division
221
(at diplotene: see Chapter 18: Male Reproductive System) so the cell is sometimes described as ‘resting’. At puberty the rat ovary contains about 40,000 primordial follicles that are transformed into primary follicles throughout the reproductive life of the animal. This transformation seems not to be under hormonal control, nor is it related to the oestrous cycle. Most transformed follicles develop for variable periods before undergoing a form of degeneration called atresia, never reaching their final preovulation state. Atresia affects follicles at all stages of development up to the antral follicle. Although it was also once thought that oogonia in the ovary gave rise to primary oocytes, this idea was discarded in the early 1950s when Zuckerman showed that the female mammal was born with a stock of primary oocytes that are used up during reproductive life and not replaced (Franchi et al., 1962; Zuckerman, 1970). Interestingly, this was the view put forward by Waldeyer as long ago as 1870, but later disputed and discarded. Zuckerman acknowledged that a few species of mammals, several prosimians in fact, did not seem to conform to his views, but these were dismissed as unimportant exceptions to the rule. The debate has been resumed in the last 20 or so years, and the question once more seems to be far from settled, though Zuckerman’s views still prevail in most textbooks (Tilly et al., 2009; Esmaeilian et al., 2013).
Primary Follicle Primary follicles can be distinguished from primordial follicles by the layer of cuboidal/columnar cells that surround the primary oocyte. These follicular cells develop further as the follicle develops.
Secondary Follicle During follicular development the primary oocyte increases in diameter, often appearing to have no nucleus; a sectioning artefact created by the oocyte being 100 µm in diameter, so there is a fair chance that a 5 µm histological section will not pass through the nucleus of the cell. During development of the secondary follicle, the follicular cells multiply and form a multilayered avascular zona glomerulosa surrounding the oocyte. The zona pellucida (pellucid means transparent) starts to appear in primary follicle, and in the secondary follicle can be clearly seen between the zona glomerulosa and the plasma membrane of the oocyte. This layer of glycoprotein and acid mucopolysaccharide stains brightly with eosin, and deeply with connective tissue stains. The origin of the zona pellucida has been debated. Fawcett (1994) stated that it was produced by the oocyte, but that contributions from the zona glomerulosa could not be excluded. What is certain is that the oocyte and the surrounding glomerulosa cells remain
222 Anatomy and Histology of the Laboratory Rat in Toxicology and Biomedical Research
in contact via gap junctions between cytoplasmic processes that pass through the zona pellucida. Another development that begins in association with the primary follicle, but which becomes increasingly clear as the follicle develops, is the condensation of stromal tissue around the developing follicle. Two distinct layers of tissue, the theca interna (or theca folliculi) and the theca externa (theca: Greek for sheath), are formed. The theca interna comprises a vascular layer of polygonal secretory cells that will play an important part in the formation of the corpus luteum if the follicle proceeds to ovulation. These cells have all the ultrastructural characteristics of steroid producing cells and are rich in smooth endoplasmic reticulum. The theca externa is a cellular connective tissue. The theca interna is separated from the outer layer of glomerulosa cells by a thick basement membrane thought to be produced by the glomerulosa cells. As the secondary follicle develops, fluid filled spaces appear among the cells of the zona glomerulosa. A follicle showing several well-developed fluid filled spaces is described as a vesicular follicle.
division, and of course prior to all mitotic divisions, DNA is duplicated. The Graafian or preovulatory follicle (named for the Dutch anatomist Regnier de Graaf, 1641 73). The follicle continues to expand and is distended with follicular fluid, reaching a diameter of 600 700 µm. The oocyte is now 80 90 µm in diameter and surrounded by a corona of glomerulosa cells that project into the follicular fluid forming a promontory known as the cumulus oophorus or discus proligerous. The root of the promontory thins and detaches from the wall of the follicle, allowing the oocyte, surrounded by its corona, to float free in the antral fluid. All is now ready for ovulation. The Graafian follicle (in the rat there will be a number of these) distends the adjacent surface of the ovary in an area called the stigma. The tunica albuginea and the OSE become thin and rather avascular; rupture of the surface occurs, and the oocyte escapes. The rupture should not be thought of as the sudden bursting of a balloon; the oocyte is released, rather than fired, from the ovary.
Tertiary or Antral Follicle
The Corpus Luteum
The tertiary or antral follicle is immediately identifiable. The fluid filled spaces of the glomerulosa coalesce to form a single cavity or antrum, while the oocyte, still surrounded by a few layers of glomerulosa cells, projects into the antrum. As the follicle develops the layers of glomerulosa cells around the oocyte decrease till just one or two layers, the corona radiate, remain. The oocyte continues to grow as the follicle develops, but has yet to progress beyond the prophase of the first meiotic division. Now things begin to move more rapidly. A number of tertiary follicles are ‘selected’ for further development into the follicles that will reach ovulation. The primary oocyte completes its first meiotic division, an unequal division producing a secondary oocyte and a smaller polar body. The secondary oocyte undergoes a second meiotic division without duplication of DNA, and then divides, producing a mature ovum and a second polar body. The first polar body can itself divide producing two further polar bodies. Polar bodies are not considered to be fertilisable and generally remain close to the oocyte, within the zona pellucida. In mammals they are short-lived, disappearing in man after 24 48 hours. Although in mammals they appear to be no more than waste bins for superfluous genetic material produced during meiosis, this is not the case for all animals. In many parthogenetic species the polar bodies are an essential part of the reproductive process. The key to understanding this rather complicated process is to recall that prior to the second meiotic division is there no duplication of DNA; prior to the first meiotic
Once the oocyte has been released, the ruptured follicle collapses in on itself. The centre fills with blood from the vascular theca interna, and the basement membrane between the theca interna and the glomerulosa breaks down. What were once the cells of the theca interna and the glomerulosa enlarge, accumulate lipid, and are transformed into plump, pale-staining polygonal cells: ‘lutein cells’ (Fawcett, 1994). Fawcett distinguished glomerulosa lutein cells from theca lutein cells, the latter being more deeply staining. The tissue of the corpus luteum is invaded by sinusoidal capillaries, becoming an endocrine gland that produces progesterone. The corpus luteum is yellowish in colour because of the lipid it contains (luteum is Latin for yellow). The further development of the corpus luteum depends on whether mating has occurred, and whether fertilisation of the ova has resulted from the mating. If no mating occurs, the corpus luteum of the rat degenerates and plays little part in suspending the oestrous cycle. It persists longer if ‘unsuccessful mating’ has occurred (such as would be produced by mating the female rat with a vasectomised male), and for much longer if fertilisation has occurred. Sterile mating leads to a state of pseudopregnancy, whereby the oestrous cycle is suspended and the phase of dioestrus prolonged. If no mating occurs, the corpus luteum regresses over the following two or three cycles. When successful mating occurs, the corpus luteum (actually corpora lutea because the rat produces more than one ovum, often eight or so) persists until the feto-placental unit takes over hormone production.
Female Reproductive Tract Chapter | 19
Complete degeneration of the corpus luteum leads to formation of a small, white, fibrous structure known as the corpora albicans, which persists in the human ovary, but disappears in the rat (Fig. 19.2).
The Oestrous Cycle of the Rat Much confusion surrounds the words oestrus and oestrous (American authors use the terms estrus and estrous). Oestrus is a noun, and means the period of sexual receptivity or ‘heat’ experienced by female rats and other mammals, but not women. Oestrous is an adjective used to describe the cycle of events occurring in the ovary, uterus and vagina of the female animal that occur before and after ovulation. Many authors do not distinguish these terms. The female rat ovulates every 4 5 days, so has a 4- to 5-day oestrous cycle. If female rats are prevented from mating this cycle will be repeated from puberty, which occurs at about 30 40 days (Porter stated 72 days but earlier opening of the vagina is usually seen) of postnatal life, until the end of reproductive life at about 450 days of postnatal life (Porter, 1957). The rat oestrous cycle has been studied in detail; see Mandl (1951). The following stages are defined: Pro-oestrus: 18 hours Oestrus: 25 hours
223
Metoestrus: 5 hours Dioestrus: 59 hours This indicated a cycle of 107 hours or 4.4 days. Slightly different periods were described by Long and Evans (1922), the earliest workers in the field. Defining where a female rat is in the cycle can be done by examining the cells by vaginal smears or by instilling and then removing fluid and examining it for cells. Five states or stages are usually defined. These are explained in Table 19.1. For a more recent description of the stages of rat oestrous, see Paccola et al. (2013). Perhaps, the most interesting aspect of the oestrous cycle: the development of the ovarian follicles and the times taken for the various stages of follicular development, is well shown in Fig. 19.3 by McGee and Hsueh (2000). Fig. 19.3 shows the slow development from the primordial follicles to the antral follicles. Once the antral follicles are recruited for further development to Graafian follicles the process runs quickly: 2 3 days in the rat. The very slow transition from primordial to antral follicles in female humans is shown for comparison. It is essential to recall that the process of transformation of primordial follicles into antral follicles is continuous but that the transformation of antral follicles into Graafian
FIGURE 19.2 Ovarian follicles: On the left hand plate a primary follicle (PR) and two primordial follicles (PM). In the centre a collection of secondary follicles (S), the larger just about to transition to being tertiary follicle, the antrum can just be seen starting to emerge. A Corpora Lutea (CL) also present. On the left hand plate a large tertiary follicle (T), with an early secondary follicle, and just visible, a primordial follicle.
TABLE 19.1 Stages of the Rat Oestrous Cycle Stage
Observations of Vagina and Cells Recovered on Swabbing
1: 12 h
Vaginal mucosa slightly dry, only epithelial cells in smear, vaginal lips a little swollen, animal coming into heat at end of stage
2:
Vaginal mucosa dry and lustreless, only cornified cells in smear, vaginal lips swollen, animal in heat
3: (stages 2 and 3: 27 h)
As in stage 2, cornified material abundant: cheesy material present. Animal not in heat. Ovulation occurs. Note that ‘heat’ is over before ovulation occurs
4: 6 h
Vaginal mucosa slightly moist, cornified cells and leucocytes in smear, swelling of vaginal lips gone
5: 57 h
Vaginal mucosa moist and glistening, leucocytes and epithelial cells in smear, variable amount of mucus present
Source: Modified from Parkes, A.S., 1929. The Internal Secretions of the Ovary. Longmans, Green and Co., London (Parkes, 1929).
224 Anatomy and Histology of the Laboratory Rat in Toxicology and Biomedical Research
Cyclic recruitment
Selection and dominance
Initial recruitment
Ovulation
Graafian follicles Secondary
Primary Primordial
Antral
human (2–5 mm) rat (0.2–0.4 mm)
Atretic
Human ??? Rat
>120 days >30 days
71 days
14 days
28 days
2–3 days
FIGURE 19.3 Duration of follicle recruitment and selection in human and rat ovaries. Primordial follicles undergo initial recruitment to enter the growing pool of primary follicles. Due to its protracted nature, the duration required for this step is unknown. In the human ovary, greater than 120 days are required for the primary follicles to reach the secondary follicle stage, whereas 71 days are needed to grow from the secondary to the early antral stage. During cyclic recruitment, increases in circulating FSH allow a cohort of antral follicles (2 5 mm in diameter) to escape apoptotic demise. Among this cohort, a leading follicle emerges as dominant by secreting high levels of estrogens and inhibins to suppress pituitary FSH release. The result is a negative selection of the remaining cohort, leading to its ultimate demise. Concomitantly, increases in local growth factors and vasculature allow a positive selection of the dominant follicle, thus ensuring its final growth and eventual ovulation. After cyclic recruitment, it takes only 2 weeks for an antral follicle to become a dominant Graafian follicle. In the rat, the duration of follicle development is much shorter than that needed for human follicles. The time required between the initial recruitment of a primordial follicle and its growth to the secondary stage is more than 30 days, whereas the time for a secondary follicle to reach the early antral stage is about 28 days. Once reaching the early antral stage (0.2 0.4 in diameter), the follicles are subjected to cyclic recruitment, and only 2 3 days needed for them to grow into preovulatory follicles. From McGee, E.A., Hsueh, A.J.W., 2000. Initial and cyclic recruitment of ovarian follicles. Endocr. Rev. 21 (2), 200 214.
follicles is cyclical and hormone-driven. Fig. 19.3 shows that some follicles, indeed most follicles, never make the full journey from primordial to Graafian, on the contrary most undergo a process of degeneration and form atretic follicles. The word atretic is derived from atresia meaning failure to penetrate or perforate; in the present case it means failure to complete development.
derivatives remain in the male and that remnants of mesonephric derivatives remain in the female. We have listed them, in Table 19.2, as they appear in humans because the reader may come across the terms in other accounts. Functional parts of the males and female systems are shown in bold type. Textbooks of human anatomy and embryology should be consulted for details of the minor structures.
Atretic Follicles
UTERUS
Atresia can occur at any point in follicular development and is typified by the apoptosis of the cells of the zona glomerulosa. Small, dark, pyknotic, nuclei appear accompanied by the nuclear debris produced by karyorrhexis (the debris is described as karyorrhectic: the adjectival form). Cells from the theca interna persist and form masses of glandular tissue, a process that is particularly marked in rodents but not in humans. The zona pellucida seems to be resistant to degeneration and spaces containing the remnants of zonae may be seen.
Rete Ovarii The remains of the mesonephric tubules sometimes appear as anastomosing tubules. Remnants of paramesonephric
The wall of the uterus may be divided into the endometrium or mucosa, the myometrium, which is the muscular layer of the wall, and the perimetrium, a thin layer of connective tissue covered by a single layer of serosal cells. The endometrium contains tubular glands that extend into the lamina propria underlying the endometrial epithelium. The endometrial epithelium is a simple columnar epithelium that varies in height with the stages of the oestrous cycle. The lamina propria is notably vascular and contains large numbers of leucocytes. The myometrium comprises an inner circular layer (with respect to the long axis of the uterus) and an outer longitudinal layer. The structure of the endometrium varies with the phases of the oestrous cycle. These changes are summarised below.
Female Reproductive Tract Chapter | 19
225
TABLE 19.2 Origins and Derivatives of the Male and Female Reproductive Systems Origin
Male Product
Female Product
Genital ridge
Testis
Ovary
Genital ridge, lower part
Gubernaculum testis
Chorda uteroinguinalis or the round ligament of the uterus
Mesonephros
Epididymis
Epoo¨phoron
Mesonephric tubules
Ductuli efferentes
Ductuli epoo¨phori transversi
Other mesonephric tubules
Ductuli aberrantes (of Halleri)
Mesonephric duct (Wolffian duct)
Paradidymis (organ of Giraldes)
Paroo¨phoron
Ductus epididymis
Ductus epoo¨phori longitudinalis
Ductus deferens
Ductus paraurethrales (of Gartner)
Ductus ejaculatorius Cystic end of mesonephric duct (derivatives described as the stalked hydatids)
Appendix epididymis (or appendix epididymidis) Sometimes called the stalked hydatid of Morgagni
Appendix vesiculosa (several may be present and are sometimes called the hydatids of Morgagni)
Paramesonephric duct (Mullerian duct)
Appendix testis (sessile hydatid, sessile hydatid of Morgagni)
Uterine tubes
Utriculus masculinus
Uterus Vagina
FIGURE 19.4 Lumen (L) is open and a mitosis can be seen in the endometrial gland marked, although they are not easy to see at this magnification (EG). The basophilic corpora lutea are large and lacking in mitoses.
Appearance of the Uterine Mucosa at Stages of the Oestrous Cycle The appearance of the corpora lutea of the ovary is shown in brackets.
can be identified among the cells of the endometrium in the early part of the stage. The lamina propria contains many polymorph inflammatory cells, increasing in number in the later stages of pro-oestrus. The glands show dilatation of their lumina (relatively high proportion of basophilic (recent) corpora lutea) (Fig. 19.4).
Pro-Oestrus The epithelium comprises medium-sized columnar cells which increase in height as the stage progresses. The lumen starts to distend with fluid and occasional mitotic figures
Oestrus The columnar cells of the epithelium reach their maximal height and as the stage progresses, apoptotic cells begin
226 Anatomy and Histology of the Laboratory Rat in Toxicology and Biomedical Research
FIGURE 19.5 In this late stage of oestrus the lumen has already collapsed but the tall columnar cells are very evident and are the extensive apoptoses in the endometrial glands. The majority of the corpora lutea are eosinophilic and some very early developing corpora lutea with high mitotic rates may be seen.
FIGURE 19.6 Apoptosis of the luminal epithelial cells now very clear to see, this generation of corpora lutea now quite large and still basophilic, with occasional mitotic figures that are quite difficult to see. The degeneration in the eosinophilic corpora lutea pictured is quite clear.
to appear in the epithelium, particularly in the endometrial glands. By the end of the stage apoptosis is significant. The lumen is fully distended with fluid. Many polymorph inflammatory cells can be seen in the lamina propria. Some endometrial glands remain dilated but others are beginning to collapse. (The ovary may have four sets of corpora lutea visible, many of which are large and eosinophilic) (Fig. 19.5).
Metoestrus Initially the columnar cells are very tall but soon reduce in height during the stage. Apoptosis is predominantly in the luminal epithelial cells, there may be an occasion apoptotic cell in the endometrial gland epithelium early on. (Three sets of corpora lutea can usually be identified and large degenerating corpora lutea prominent.) (Fig. 19.6).
Dioestrus The height of the columnar cells and that of the cells of the glandular epithelium is reduced to a minimum level. Fewer inflammatory cells are seen in the lamina propria but the fibroblasts of the lamina propria are plump as a result of activation by progesterone produced by the corpus luteum. Mitotic figures in endometrial cells begin to appear in early dioestrus and increase with time and are probably most common in the second half of the stage. (Corpora lutea become more eosinophilic as the cycle progresses so mostly eosinophilic corpora lutea as the ovary enters proestrus) (Fig. 19.7). Though the changes just described can be identified at microscopy they are, of course, much less marked that those occurring in the female human in whom the lining of the uterus undergoes spectacular degeneration and is shed with the loss of a considerable volume of blood.
Female Reproductive Tract Chapter | 19
227
FIGURE 19.7 Quiescent appearance of the uterus in the main, although one or two mitotic figures can be seen in the luminal epithelium. The corpora luteal cells in this generation of corpora lutea are now quite plump and increasingly eosinophilic.
FIGURE 19.8 Proestrus to the left and oestrus to the right. Both stages are quite easy to differentiate.
These marked changes are the hallmark of the menstrual cycle and are seen only in primates.
VAGINA The vagina of the adult rat is about 15 20 mm in length and about 3 5 mm in diameter when distended (Hebel and Stromberg, 1976). The wall has a mucosa and a muscular layer. The mucosa has a stratified squamous epithelium that varies in structure with the stage of the oestrous cycle. Rather unusually for a stratified squamous epithelium, the cells and the surface cells of the epithelium produce mucus at one stage of the oestrous cycle, but at another stage undergo cornification. During the production of mucus, the surface layers of the epithelium are described as the stratum mucification, and as the stratum corneum during the cornification stage. When fully developed, the cells of the stratum mucification lie on the
stratum corneum. The deeper layers of the epithelium comprise the stratum germinativum, stratum spinosum and stratum granulosum, as seen in other stratified squamous epithelia of the keratinising type. Hebel and Stromberg (1976) noted that during the cornification stage a well-defined stratum lucidum could be identified. The muscle of the wall is largely longitudinal in arrangement. These changes are summarised below:
Appearance of the Epithelium at Stages of the Oestrous Cycle Pro-Oestrus Stratum mucification present. Stratum corneum appears at the surface during later pro-oestrus. Occasional polymorphs present in epithelium.
228 Anatomy and Histology of the Laboratory Rat in Toxicology and Biomedical Research
FIGURE 19.9 Metoestrus and dioestrus. Much more difficult to differentiate. Top left is early metoestrus and top right late metoestrus. Bottom frames early and late dioestrus.
Oestrus
Notes on Cycle Staging
Stratum mucification not present. Stratum corneum well developed and cornified cells appear in the lumen of the vagina. Polymorphs increasing in number (Fig. 19.8).
Staging the oestrus cycle is considerably more straightforward than staging spermatogenic cycles in the testes. There are however a few recommendations that are worthy of consideration before beginning.
Metoestrus
1. Take longitudinal sections of the vagina, the changes in the female reproductive tract pass though in waves and not all parts will be the same at any one time. 2. Be wary of cage effects, females in the same cage tend to synchronise their cycling. 3. Given the small numbers of animals normally used in most studies (10 or less), and the variation you see as background, it is unlikely you will see an effect in terms of the numbers of animals in different stages of the cycle, but asynchronicity of cycling (vagina, uterus and ovaries appear to be indifferent stages) is a very sensitive indicator of endocrine disruption.
Stratum mucification, stratum corneum and stratum granulosum absent. Stratum germinativum reduced to four to six layers of cells. The many polymorphs present early in metoestrus later decline in number.
Dioestrus Stratum germinativum shows plump, polygonal cells: this change is described as early mucification of the stratum germinativum. Few polymorphs present (Fig. 19.9).
Female Reproductive Tract Chapter | 19
4. Always remember that it is a continuous cycle and there will be grey areas. 5. Your counts should be roughly in line with the length of each stage. If they are not, you are probably making a mistake. Most common error is to over report metoestrus.
REFERENCES Auersperg, N., Wong, A.S.T., Choi, K.-C., Kang, S.K., Leung, P.C.K., 2001. Ovarian surface epithelium: biology, endocrinology and pathology. Endocr. Rev. 22 (2), 255 288. Esmaeilian, Y., Atalay, A., Erdemli, E., 2015. Post-natal oogenesis: a concept for controversy that intensified during the last decade. Zygote. 23, 315 326. Fawcett, D.W., 1994. A Textbook of Histology, twelfth ed. Chapman & Hall, New York and London. Franchi, L.L., Mandl, A.M., Zuckerman, S., 1962. The development of the ovary and the process of oogenesis (Chapter 1) In: Zuckerman, S., Mandl, A.M., Eckstein, P. (Eds.), The Ovary, 2 Volumes. Academic press, New York and London. Gayta´n, M., Sa´nchez, M.A., Morales, C., Bellido, C., Milla´n, Y., de las Mulas, J.M., et al., 2005. Cyclic changes of the ovarian surface epithelium in the rat. Reproduction. 129, 311 321. Hebel, R., Stromberg, M.W., 1976. Anatomy of the Laboratory Rat. The Williams & Wilkins Company, Baltimore, MD. Hunt, H.R., 1924. A Laboratory Manual of the Anatomy of the Rat. The Macmillan Company, New York. Long, J.A., Evans, H.M., 1922. The oestrous cycle in the rat and its associated phenomena. Mem. Univ. Calif. 6, 1 148. Mandl, A.M., 1951. The phases of the oestrous cycle in the adult white rat. J. Exp. Biol. 28, 576 584. McGee, E.A., Hsueh, A.J.W., 2000. Initial and cyclic recruitment of ovarian follicles. Endocr. Rev. 21 (2), 200 214. Paccola, C.C., Resende, C.G., Stumpp, T., Miraglia, S.M., Cipriano, I., 2013. The rat estrous cycle revisited: a quantitative and qualitative analysis. Anim. Reprod. 10 (4), 677 683.
229
Parkes, A.S., 1929. The Internal Secretions of the Ovary. Longmans, Green and Co, London. Porter, G., 1957. The Norway Rat (Chapter 32) In: Worden, A.N., LanePetter, W. (Eds.), The UFAW Handbook on the Care and Management of Laboratory Animals, second ed. Universities Federation for Animal Welfare, London, pp. 344 377. Rowett, H.G.Q., 1952. Dissection Guides: III The Rat (With Notes on the Mouse). John Murray, London. Tilly, J.L., Niikura, Y., Rueda, B.R., 2009. The current status of evidence for and against postnatal oogenesis in mammals: a case of ovarian optimism versus pessimism? Biol. Reprod. 80, 2 12. Van der Schoot, P., Emmen, J.M.A., 1996. Development, structure and function of the cranial suspensory ligaments of the mammalian gonads in a cross-species perspective; their possible role in effecting disturbed testicular function. Hum. Reprod. Update. 2 (5), 399 418. Waldeyer, W., 1870. Eierstock und Ei. Engelmann, Leipzig. Zuckerman, S., 1970. A story about mammalian egg-cells (Chapter 3, pp. 22 34) In: Zuckerman, S. (Ed.), Beyond the Ivory Tower. Weidenfeld and Nicolson, London.
FURTHER READING Parkes, A.S. Marshall’s Physiology of Reproduction. third ed. Longmans, Green and Co., London, New York, Toronto. Volume 1, part one: 1956; Volume 1, part two: 1960; Volume 2: 1952; Volume 3: 1966. (Irreplaceable as an account of the comparative anatomy of the reproductive system and of the physiology as understood at the time it was written. A Fourth Edition was produced: possibly incomplete). Zuckerman, S., Mandl, A.M., Eckstein, P., 1962. The Ovary. 2 Volumes. Academic press, New York and London (Invaluable for details of comparative anatomy).
Female Reproductive Tract The female reproductive tract comprises the ovaries, oviducts, the uterus and the vagina. Although the rat cervix is not comparable to that found in humans, the junction between the uterus and the vagina is sometimes referred to as the cervix.
DEVELOPMENT AND TOPOGRAPHY The first stages of the development of the ovary are identical to those of the testis, and cannot be distinguished histologically; the later stages are very different. The ovary remains within the abdominal cavity, while the testis descends to the scrotum. The oviducts and uterus are formed from the paramesonephric ducts (Mu¨llerian ducts), which develop from the lining of the peritoneal cavity close to the mesonephric ducts (Wolffian ducts), but have nothing to do with the ‘second kidney’ or mesonephros (see Chapter 18: Male Reproductive System). The paramesonephric ducts are attached to the dorsal abdominal wall by a fold of parietal peritoneum, which develops into the broad ligament of the uterus. The ovary is attached to the surface of the anterior end of the broad ligament by a band of connective tissue known as the mesovarium. In humans, ova released from these surfaces enter the peritoneal cavity, but are quickly swept into the funnel-shaped mouths of the oviducts that run in the broad ligament and open into the peritoneal cavity, very close to the ovary. The oviduct is sometimes called the salpinx (Greek for tube), and the part of the broad ligament that enfolds the salpinx is the mesosalpinx. It will probably be obvious that there is a danger of infection making its way via the vagina, uterus and oviducts into the peritoneal cavity. Infection of the oviduct is referred to as salpingitis, and is one of the most common causes of infertility in women. The anatomy of the rat is a little different, as the ovary is surrounded by a capsule of mesovarium, the ovarian bursa, pierced by the oviduct. The open end of the oviduct lies within it and the ova never actually enter the peritoneal cavity. This arrangement is unusual among
mammals, and some authors maintain that the capsule around the rat ovary is not complete and that a slit connects the interior of the capsule with the peritoneal cavity. The developing ovary, like the developing testis, is attached to the gubernaculum, which itself is attached to the developing uterus. The gubernaculum develops into the round ligament of the ovary, which connects the ovary with the tip of the adjacent uterine horn and the round ligament of the uterus, which connects the uterus with the abdominal wall at the internal inguinal ring. In Chapter 18, Male Reproductive System, we describe how the testis, accompanied by a projection of parietal peritoneum (the processus vaginalis), passes through the inguinal canal and into the scrotum. Things are not so very different in the female. The round ligament of the uterus, accompanied for some distance by a female equivalent of the processus vaginalis (human anatomists call this female equivalent the canal of Nuck), passes through the inguinal canal and into the labia majora of the external genitalia. Although this is what happens in female humans, it would seem that diverticuli of the peritoneum (the equivalents of the processus vaginalis) are not always present in female rats (Hunt, 1924). Hebel and Stromberg (1976) refer to the ligament of the ovary that is derived from the gubernaculum as the ‘proper ligament of the ovary’, placed it medial to the oviduct, and described it as blending with the mesosalpinx at the tip of the uterine horn. This ligament contains mainly smooth muscle fibres. They also describe a suspensory ligament that runs from the ovarian hilum to the crus of the diaphragm. This is presumably equivalent to the suspensory ligament of the human ovary (better described in human anatomy as the infundibulopelvic ligament) that forms that part of the broad ligament running from the ovary to the pelvic wall. Der Schoot and Emmen (1996) have provided an interesting discussion of the suspensory ligaments of mammalian gonads. In humans, the ovary descends to the brim of the pelvis, but in the rat, the two horns of the uterus stretch forwards to the ovaries situated on the dorsal aspect of the abdominal wall.
Anatomy and Histology of the Laboratory Rat in Toxicology and Biomedical Research. DOI: https://doi.org/10.1016/B978-0-12-811837-5.00019-8 © 2019 Elsevier Inc. All rights reserved.
219
220 Anatomy and Histology of the Laboratory Rat in Toxicology and Biomedical Research
The Ovary The ovaries of the rat lie caudal to the kidneys. Hebel and Stromberg (1976) placed the right ovary at the level of lumbar vertebrae 4 5 and the left at 5 6, but Rowett (1952) showed them a little further forward, with the left lying alongside the left kidney (Fig. 19.1). In the mature adult, the ovary measures a 5 3 4 3 3 mm3 and weighs about 60 mg. Blood is supplied to the right ovary by a branch of the abdominal aorta, the right ovarian artery and returned via the right ovarian vein to the posterior vena cava. On the left, the vessels are connected with the renal vessels. The ovarian arteries run in the broad ligament and anastomose with the uterine arteries, which arise from the iliac arteries.
Oviduct The oviduct is a narrow looped structure, 0.4 0.5 mm in diameter and 18 30 mm in length, lying adjacent to the ovary, with a funnel-shaped proximal end that pierces the ovarian bursa (Hebel and Stromberg, 1976). The distal end penetrates the tip of the uterus. The mucosa of the proximal part of the oviduct is thrown into a series of branching longitudinal folds, which in cross section appear to almost fill the lumen, extending beyond the open cranial end of the oviduct as finger-like
processes called fimbria. The folds become less prominent as the uterus is approached. The mucosa is a simple columnar epithelium, with both ciliated and unciliated secretory cells. The ciliated cells are said to respond to oestrogen by increasing the frequency of ciliary beating at the time of ovulation. Beneath the epithelium a very vascular lamina propria contains large lymphatic capillaries. There is little evidence of a muscularis mucosae, though some smooth muscle fibres extend into the longitudinal folds. The wall of the oviduct contains a well-developed circular or spiral layer smooth muscle (the muscularis), and a less welldeveloped outer layer of longitudinally disposed smooth muscle. Rhythmic pumping of the tube aids transport of the fertilised ovum to the uterus. It is important to note that fertilisation, the entry of a spermatozoon into the ovum, occurs in the oviduct. The wall of the oviduct is very vascular with a well-developed layer of blood vessels between the circular and longitudinal layers of smooth muscle.
Uterus The rat has a duplex uterus that is it is divided into two separate horns. The horns are 30 40 mm in length and join about 7 10 mm above the vagina, but each horn enters the vagina individually, so functionally the rat can FIGURE 19.1 Dissection to show female reproductive system.
Adrenal gland Fat Kidney Ovary
Ureter
Uterus Mesovarium of oviduct
Bladder Vagina Cut pelvic girdle Vagina Anus Clitoris
Female Reproductive Tract Chapter | 19
be considered to have two uteri. Each opening of a uterine horn into the vagina is surrounded by a small ventral cushion, a large dorsal cushion and two lateral cushions of tissue. Hebel and Stromberg (1976) refer to this as the ‘portio vaginalis uteri’, the doorway from the uterus into the vagina. A short sagittal partition separates the entry points of the horns into the vagina.
Vagina The rat vagina opens to the exterior just in front of the anus and behind the opening of the urethra. The vagina is thin walled, 15 20 mm in length and 3 5 mm in diameter, with a mucosa that is thrown into longitudinal folds.
HISTOLOGY The surface of the ovary is covered by a simple epithelium, once called the germinal epithelium as it was thought that it was the source of the germ cells. This concept has long since been abandoned, and the epithelium is now called the ovarian surface epithelium (OSE). It is derived from the lining of the peritoneal cavity and is really modified parietal peritoneum. The cells of the epithelium vary from squamous to cuboidal, and play an important part in repairing the breach made in the surface of the ovary when ovulation occurs. The OSE has attracted much interest during the past 30 or so years because .80% of human ovarian cancers arise from this epithelium. The OSE has been reviewed in detail by Auersperg et al. (2001) and with regard to the rat by Gayta´n et al. (2005). Beneath the OSE lies the tunica albuginea, a layer of connective tissue containing many fibroblast-like cells. The collagen fibres of the tunica are thin and less well developed than those of the testes. The remainder of the ovary can be divided into a cortex and a medulla. The cortex contains the follicles, the medulla and the large blood vessels running to and from the ovary at the hilum. Between the follicles of the cortex and the blood vessels of the medulla is the ovarian stroma, a fibroblast-rich connective tissue.
Follicles Primordial Follicle Immediately beneath the tunica albuginea lie the primordial follicles. These comprise a primary oocyte surrounded by a single layer of squamous follicular cells. The oocyte is about 25 µm in diameter with a welldefined nucleus, and a nucleolus that often stains pink in H&E stained sections. The development of the primary oocyte arrests during prophase of the first meiotic division
221
(at diplotene: see Chapter 18: Male Reproductive System) so the cell is sometimes described as ‘resting’. At puberty the rat ovary contains about 40,000 primordial follicles that are transformed into primary follicles throughout the reproductive life of the animal. This transformation seems not to be under hormonal control, nor is it related to the oestrous cycle. Most transformed follicles develop for variable periods before undergoing a form of degeneration called atresia, never reaching their final preovulation state. Atresia affects follicles at all stages of development up to the antral follicle. Although it was also once thought that oogonia in the ovary gave rise to primary oocytes, this idea was discarded in the early 1950s when Zuckerman showed that the female mammal was born with a stock of primary oocytes that are used up during reproductive life and not replaced (Franchi et al., 1962; Zuckerman, 1970). Interestingly, this was the view put forward by Waldeyer as long ago as 1870, but later disputed and discarded. Zuckerman acknowledged that a few species of mammals, several prosimians in fact, did not seem to conform to his views, but these were dismissed as unimportant exceptions to the rule. The debate has been resumed in the last 20 or so years, and the question once more seems to be far from settled, though Zuckerman’s views still prevail in most textbooks (Tilly et al., 2009; Esmaeilian et al., 2013).
Primary Follicle Primary follicles can be distinguished from primordial follicles by the layer of cuboidal/columnar cells that surround the primary oocyte. These follicular cells develop further as the follicle develops.
Secondary Follicle During follicular development the primary oocyte increases in diameter, often appearing to have no nucleus; a sectioning artefact created by the oocyte being 100 µm in diameter, so there is a fair chance that a 5 µm histological section will not pass through the nucleus of the cell. During development of the secondary follicle, the follicular cells multiply and form a multilayered avascular zona glomerulosa surrounding the oocyte. The zona pellucida (pellucid means transparent) starts to appear in primary follicle, and in the secondary follicle can be clearly seen between the zona glomerulosa and the plasma membrane of the oocyte. This layer of glycoprotein and acid mucopolysaccharide stains brightly with eosin, and deeply with connective tissue stains. The origin of the zona pellucida has been debated. Fawcett (1994) stated that it was produced by the oocyte, but that contributions from the zona glomerulosa could not be excluded. What is certain is that the oocyte and the surrounding glomerulosa cells remain
222 Anatomy and Histology of the Laboratory Rat in Toxicology and Biomedical Research
in contact via gap junctions between cytoplasmic processes that pass through the zona pellucida. Another development that begins in association with the primary follicle, but which becomes increasingly clear as the follicle develops, is the condensation of stromal tissue around the developing follicle. Two distinct layers of tissue, the theca interna (or theca folliculi) and the theca externa (theca: Greek for sheath), are formed. The theca interna comprises a vascular layer of polygonal secretory cells that will play an important part in the formation of the corpus luteum if the follicle proceeds to ovulation. These cells have all the ultrastructural characteristics of steroid producing cells and are rich in smooth endoplasmic reticulum. The theca externa is a cellular connective tissue. The theca interna is separated from the outer layer of glomerulosa cells by a thick basement membrane thought to be produced by the glomerulosa cells. As the secondary follicle develops, fluid filled spaces appear among the cells of the zona glomerulosa. A follicle showing several well-developed fluid filled spaces is described as a vesicular follicle.
division, and of course prior to all mitotic divisions, DNA is duplicated. The Graafian or preovulatory follicle (named for the Dutch anatomist Regnier de Graaf, 1641 73). The follicle continues to expand and is distended with follicular fluid, reaching a diameter of 600 700 µm. The oocyte is now 80 90 µm in diameter and surrounded by a corona of glomerulosa cells that project into the follicular fluid forming a promontory known as the cumulus oophorus or discus proligerous. The root of the promontory thins and detaches from the wall of the follicle, allowing the oocyte, surrounded by its corona, to float free in the antral fluid. All is now ready for ovulation. The Graafian follicle (in the rat there will be a number of these) distends the adjacent surface of the ovary in an area called the stigma. The tunica albuginea and the OSE become thin and rather avascular; rupture of the surface occurs, and the oocyte escapes. The rupture should not be thought of as the sudden bursting of a balloon; the oocyte is released, rather than fired, from the ovary.
Tertiary or Antral Follicle
The Corpus Luteum
The tertiary or antral follicle is immediately identifiable. The fluid filled spaces of the glomerulosa coalesce to form a single cavity or antrum, while the oocyte, still surrounded by a few layers of glomerulosa cells, projects into the antrum. As the follicle develops the layers of glomerulosa cells around the oocyte decrease till just one or two layers, the corona radiate, remain. The oocyte continues to grow as the follicle develops, but has yet to progress beyond the prophase of the first meiotic division. Now things begin to move more rapidly. A number of tertiary follicles are ‘selected’ for further development into the follicles that will reach ovulation. The primary oocyte completes its first meiotic division, an unequal division producing a secondary oocyte and a smaller polar body. The secondary oocyte undergoes a second meiotic division without duplication of DNA, and then divides, producing a mature ovum and a second polar body. The first polar body can itself divide producing two further polar bodies. Polar bodies are not considered to be fertilisable and generally remain close to the oocyte, within the zona pellucida. In mammals they are short-lived, disappearing in man after 24 48 hours. Although in mammals they appear to be no more than waste bins for superfluous genetic material produced during meiosis, this is not the case for all animals. In many parthogenetic species the polar bodies are an essential part of the reproductive process. The key to understanding this rather complicated process is to recall that prior to the second meiotic division is there no duplication of DNA; prior to the first meiotic
Once the oocyte has been released, the ruptured follicle collapses in on itself. The centre fills with blood from the vascular theca interna, and the basement membrane between the theca interna and the glomerulosa breaks down. What were once the cells of the theca interna and the glomerulosa enlarge, accumulate lipid, and are transformed into plump, pale-staining polygonal cells: ‘lutein cells’ (Fawcett, 1994). Fawcett distinguished glomerulosa lutein cells from theca lutein cells, the latter being more deeply staining. The tissue of the corpus luteum is invaded by sinusoidal capillaries, becoming an endocrine gland that produces progesterone. The corpus luteum is yellowish in colour because of the lipid it contains (luteum is Latin for yellow). The further development of the corpus luteum depends on whether mating has occurred, and whether fertilisation of the ova has resulted from the mating. If no mating occurs, the corpus luteum of the rat degenerates and plays little part in suspending the oestrous cycle. It persists longer if ‘unsuccessful mating’ has occurred (such as would be produced by mating the female rat with a vasectomised male), and for much longer if fertilisation has occurred. Sterile mating leads to a state of pseudopregnancy, whereby the oestrous cycle is suspended and the phase of dioestrus prolonged. If no mating occurs, the corpus luteum regresses over the following two or three cycles. When successful mating occurs, the corpus luteum (actually corpora lutea because the rat produces more than one ovum, often eight or so) persists until the feto-placental unit takes over hormone production.
Female Reproductive Tract Chapter | 19
Complete degeneration of the corpus luteum leads to formation of a small, white, fibrous structure known as the corpora albicans, which persists in the human ovary, but disappears in the rat (Fig. 19.2).
The Oestrous Cycle of the Rat Much confusion surrounds the words oestrus and oestrous (American authors use the terms estrus and estrous). Oestrus is a noun, and means the period of sexual receptivity or ‘heat’ experienced by female rats and other mammals, but not women. Oestrous is an adjective used to describe the cycle of events occurring in the ovary, uterus and vagina of the female animal that occur before and after ovulation. Many authors do not distinguish these terms. The female rat ovulates every 4 5 days, so has a 4- to 5-day oestrous cycle. If female rats are prevented from mating this cycle will be repeated from puberty, which occurs at about 30 40 days (Porter stated 72 days but earlier opening of the vagina is usually seen) of postnatal life, until the end of reproductive life at about 450 days of postnatal life (Porter, 1957). The rat oestrous cycle has been studied in detail; see Mandl (1951). The following stages are defined: Pro-oestrus: 18 hours Oestrus: 25 hours
223
Metoestrus: 5 hours Dioestrus: 59 hours This indicated a cycle of 107 hours or 4.4 days. Slightly different periods were described by Long and Evans (1922), the earliest workers in the field. Defining where a female rat is in the cycle can be done by examining the cells by vaginal smears or by instilling and then removing fluid and examining it for cells. Five states or stages are usually defined. These are explained in Table 19.1. For a more recent description of the stages of rat oestrous, see Paccola et al. (2013). Perhaps, the most interesting aspect of the oestrous cycle: the development of the ovarian follicles and the times taken for the various stages of follicular development, is well shown in Fig. 19.3 by McGee and Hsueh (2000). Fig. 19.3 shows the slow development from the primordial follicles to the antral follicles. Once the antral follicles are recruited for further development to Graafian follicles the process runs quickly: 2 3 days in the rat. The very slow transition from primordial to antral follicles in female humans is shown for comparison. It is essential to recall that the process of transformation of primordial follicles into antral follicles is continuous but that the transformation of antral follicles into Graafian
FIGURE 19.2 Ovarian follicles: On the left hand plate a primary follicle (PR) and two primordial follicles (PM). In the centre a collection of secondary follicles (S), the larger just about to transition to being tertiary follicle, the antrum can just be seen starting to emerge. A Corpora Lutea (CL) also present. On the left hand plate a large tertiary follicle (T), with an early secondary follicle, and just visible, a primordial follicle.
TABLE 19.1 Stages of the Rat Oestrous Cycle Stage
Observations of Vagina and Cells Recovered on Swabbing
1: 12 h
Vaginal mucosa slightly dry, only epithelial cells in smear, vaginal lips a little swollen, animal coming into heat at end of stage
2:
Vaginal mucosa dry and lustreless, only cornified cells in smear, vaginal lips swollen, animal in heat
3: (stages 2 and 3: 27 h)
As in stage 2, cornified material abundant: cheesy material present. Animal not in heat. Ovulation occurs. Note that ‘heat’ is over before ovulation occurs
4: 6 h
Vaginal mucosa slightly moist, cornified cells and leucocytes in smear, swelling of vaginal lips gone
5: 57 h
Vaginal mucosa moist and glistening, leucocytes and epithelial cells in smear, variable amount of mucus present
Source: Modified from Parkes, A.S., 1929. The Internal Secretions of the Ovary. Longmans, Green and Co., London (Parkes, 1929).
224 Anatomy and Histology of the Laboratory Rat in Toxicology and Biomedical Research
Cyclic recruitment
Selection and dominance
Initial recruitment
Ovulation
Graafian follicles Secondary
Primary Primordial
Antral
human (2–5 mm) rat (0.2–0.4 mm)
Atretic
Human ??? Rat
>120 days >30 days
71 days
14 days
28 days
2–3 days
FIGURE 19.3 Duration of follicle recruitment and selection in human and rat ovaries. Primordial follicles undergo initial recruitment to enter the growing pool of primary follicles. Due to its protracted nature, the duration required for this step is unknown. In the human ovary, greater than 120 days are required for the primary follicles to reach the secondary follicle stage, whereas 71 days are needed to grow from the secondary to the early antral stage. During cyclic recruitment, increases in circulating FSH allow a cohort of antral follicles (2 5 mm in diameter) to escape apoptotic demise. Among this cohort, a leading follicle emerges as dominant by secreting high levels of estrogens and inhibins to suppress pituitary FSH release. The result is a negative selection of the remaining cohort, leading to its ultimate demise. Concomitantly, increases in local growth factors and vasculature allow a positive selection of the dominant follicle, thus ensuring its final growth and eventual ovulation. After cyclic recruitment, it takes only 2 weeks for an antral follicle to become a dominant Graafian follicle. In the rat, the duration of follicle development is much shorter than that needed for human follicles. The time required between the initial recruitment of a primordial follicle and its growth to the secondary stage is more than 30 days, whereas the time for a secondary follicle to reach the early antral stage is about 28 days. Once reaching the early antral stage (0.2 0.4 in diameter), the follicles are subjected to cyclic recruitment, and only 2 3 days needed for them to grow into preovulatory follicles. From McGee, E.A., Hsueh, A.J.W., 2000. Initial and cyclic recruitment of ovarian follicles. Endocr. Rev. 21 (2), 200 214.
follicles is cyclical and hormone-driven. Fig. 19.3 shows that some follicles, indeed most follicles, never make the full journey from primordial to Graafian, on the contrary most undergo a process of degeneration and form atretic follicles. The word atretic is derived from atresia meaning failure to penetrate or perforate; in the present case it means failure to complete development.
derivatives remain in the male and that remnants of mesonephric derivatives remain in the female. We have listed them, in Table 19.2, as they appear in humans because the reader may come across the terms in other accounts. Functional parts of the males and female systems are shown in bold type. Textbooks of human anatomy and embryology should be consulted for details of the minor structures.
Atretic Follicles
UTERUS
Atresia can occur at any point in follicular development and is typified by the apoptosis of the cells of the zona glomerulosa. Small, dark, pyknotic, nuclei appear accompanied by the nuclear debris produced by karyorrhexis (the debris is described as karyorrhectic: the adjectival form). Cells from the theca interna persist and form masses of glandular tissue, a process that is particularly marked in rodents but not in humans. The zona pellucida seems to be resistant to degeneration and spaces containing the remnants of zonae may be seen.
Rete Ovarii The remains of the mesonephric tubules sometimes appear as anastomosing tubules. Remnants of paramesonephric
The wall of the uterus may be divided into the endometrium or mucosa, the myometrium, which is the muscular layer of the wall, and the perimetrium, a thin layer of connective tissue covered by a single layer of serosal cells. The endometrium contains tubular glands that extend into the lamina propria underlying the endometrial epithelium. The endometrial epithelium is a simple columnar epithelium that varies in height with the stages of the oestrous cycle. The lamina propria is notably vascular and contains large numbers of leucocytes. The myometrium comprises an inner circular layer (with respect to the long axis of the uterus) and an outer longitudinal layer. The structure of the endometrium varies with the phases of the oestrous cycle. These changes are summarised below.
Female Reproductive Tract Chapter | 19
225
TABLE 19.2 Origins and Derivatives of the Male and Female Reproductive Systems Origin
Male Product
Female Product
Genital ridge
Testis
Ovary
Genital ridge, lower part
Gubernaculum testis
Chorda uteroinguinalis or the round ligament of the uterus
Mesonephros
Epididymis
Epoo¨phoron
Mesonephric tubules
Ductuli efferentes
Ductuli epoo¨phori transversi
Other mesonephric tubules
Ductuli aberrantes (of Halleri)
Mesonephric duct (Wolffian duct)
Paradidymis (organ of Giraldes)
Paroo¨phoron
Ductus epididymis
Ductus epoo¨phori longitudinalis
Ductus deferens
Ductus paraurethrales (of Gartner)
Ductus ejaculatorius Cystic end of mesonephric duct (derivatives described as the stalked hydatids)
Appendix epididymis (or appendix epididymidis) Sometimes called the stalked hydatid of Morgagni
Appendix vesiculosa (several may be present and are sometimes called the hydatids of Morgagni)
Paramesonephric duct (Mullerian duct)
Appendix testis (sessile hydatid, sessile hydatid of Morgagni)
Uterine tubes
Utriculus masculinus
Uterus Vagina
FIGURE 19.4 Lumen (L) is open and a mitosis can be seen in the endometrial gland marked, although they are not easy to see at this magnification (EG). The basophilic corpora lutea are large and lacking in mitoses.
Appearance of the Uterine Mucosa at Stages of the Oestrous Cycle The appearance of the corpora lutea of the ovary is shown in brackets.
can be identified among the cells of the endometrium in the early part of the stage. The lamina propria contains many polymorph inflammatory cells, increasing in number in the later stages of pro-oestrus. The glands show dilatation of their lumina (relatively high proportion of basophilic (recent) corpora lutea) (Fig. 19.4).
Pro-Oestrus The epithelium comprises medium-sized columnar cells which increase in height as the stage progresses. The lumen starts to distend with fluid and occasional mitotic figures
Oestrus The columnar cells of the epithelium reach their maximal height and as the stage progresses, apoptotic cells begin
226 Anatomy and Histology of the Laboratory Rat in Toxicology and Biomedical Research
FIGURE 19.5 In this late stage of oestrus the lumen has already collapsed but the tall columnar cells are very evident and are the extensive apoptoses in the endometrial glands. The majority of the corpora lutea are eosinophilic and some very early developing corpora lutea with high mitotic rates may be seen.
FIGURE 19.6 Apoptosis of the luminal epithelial cells now very clear to see, this generation of corpora lutea now quite large and still basophilic, with occasional mitotic figures that are quite difficult to see. The degeneration in the eosinophilic corpora lutea pictured is quite clear.
to appear in the epithelium, particularly in the endometrial glands. By the end of the stage apoptosis is significant. The lumen is fully distended with fluid. Many polymorph inflammatory cells can be seen in the lamina propria. Some endometrial glands remain dilated but others are beginning to collapse. (The ovary may have four sets of corpora lutea visible, many of which are large and eosinophilic) (Fig. 19.5).
Metoestrus Initially the columnar cells are very tall but soon reduce in height during the stage. Apoptosis is predominantly in the luminal epithelial cells, there may be an occasion apoptotic cell in the endometrial gland epithelium early on. (Three sets of corpora lutea can usually be identified and large degenerating corpora lutea prominent.) (Fig. 19.6).
Dioestrus The height of the columnar cells and that of the cells of the glandular epithelium is reduced to a minimum level. Fewer inflammatory cells are seen in the lamina propria but the fibroblasts of the lamina propria are plump as a result of activation by progesterone produced by the corpus luteum. Mitotic figures in endometrial cells begin to appear in early dioestrus and increase with time and are probably most common in the second half of the stage. (Corpora lutea become more eosinophilic as the cycle progresses so mostly eosinophilic corpora lutea as the ovary enters proestrus) (Fig. 19.7). Though the changes just described can be identified at microscopy they are, of course, much less marked that those occurring in the female human in whom the lining of the uterus undergoes spectacular degeneration and is shed with the loss of a considerable volume of blood.
Female Reproductive Tract Chapter | 19
227
FIGURE 19.7 Quiescent appearance of the uterus in the main, although one or two mitotic figures can be seen in the luminal epithelium. The corpora luteal cells in this generation of corpora lutea are now quite plump and increasingly eosinophilic.
FIGURE 19.8 Proestrus to the left and oestrus to the right. Both stages are quite easy to differentiate.
These marked changes are the hallmark of the menstrual cycle and are seen only in primates.
VAGINA The vagina of the adult rat is about 15 20 mm in length and about 3 5 mm in diameter when distended (Hebel and Stromberg, 1976). The wall has a mucosa and a muscular layer. The mucosa has a stratified squamous epithelium that varies in structure with the stage of the oestrous cycle. Rather unusually for a stratified squamous epithelium, the cells and the surface cells of the epithelium produce mucus at one stage of the oestrous cycle, but at another stage undergo cornification. During the production of mucus, the surface layers of the epithelium are described as the stratum mucification, and as the stratum corneum during the cornification stage. When fully developed, the cells of the stratum mucification lie on the
stratum corneum. The deeper layers of the epithelium comprise the stratum germinativum, stratum spinosum and stratum granulosum, as seen in other stratified squamous epithelia of the keratinising type. Hebel and Stromberg (1976) noted that during the cornification stage a well-defined stratum lucidum could be identified. The muscle of the wall is largely longitudinal in arrangement. These changes are summarised below:
Appearance of the Epithelium at Stages of the Oestrous Cycle Pro-Oestrus Stratum mucification present. Stratum corneum appears at the surface during later pro-oestrus. Occasional polymorphs present in epithelium.
228 Anatomy and Histology of the Laboratory Rat in Toxicology and Biomedical Research
FIGURE 19.9 Metoestrus and dioestrus. Much more difficult to differentiate. Top left is early metoestrus and top right late metoestrus. Bottom frames early and late dioestrus.
Oestrus
Notes on Cycle Staging
Stratum mucification not present. Stratum corneum well developed and cornified cells appear in the lumen of the vagina. Polymorphs increasing in number (Fig. 19.8).
Staging the oestrus cycle is considerably more straightforward than staging spermatogenic cycles in the testes. There are however a few recommendations that are worthy of consideration before beginning.
Metoestrus
1. Take longitudinal sections of the vagina, the changes in the female reproductive tract pass though in waves and not all parts will be the same at any one time. 2. Be wary of cage effects, females in the same cage tend to synchronise their cycling. 3. Given the small numbers of animals normally used in most studies (10 or less), and the variation you see as background, it is unlikely you will see an effect in terms of the numbers of animals in different stages of the cycle, but asynchronicity of cycling (vagina, uterus and ovaries appear to be indifferent stages) is a very sensitive indicator of endocrine disruption.
Stratum mucification, stratum corneum and stratum granulosum absent. Stratum germinativum reduced to four to six layers of cells. The many polymorphs present early in metoestrus later decline in number.
Dioestrus Stratum germinativum shows plump, polygonal cells: this change is described as early mucification of the stratum germinativum. Few polymorphs present (Fig. 19.9).
Female Reproductive Tract Chapter | 19
4. Always remember that it is a continuous cycle and there will be grey areas. 5. Your counts should be roughly in line with the length of each stage. If they are not, you are probably making a mistake. Most common error is to over report metoestrus.
REFERENCES Auersperg, N., Wong, A.S.T., Choi, K.-C., Kang, S.K., Leung, P.C.K., 2001. Ovarian surface epithelium: biology, endocrinology and pathology. Endocr. Rev. 22 (2), 255 288. Esmaeilian, Y., Atalay, A., Erdemli, E., 2015. Post-natal oogenesis: a concept for controversy that intensified during the last decade. Zygote. 23, 315 326. Fawcett, D.W., 1994. A Textbook of Histology, twelfth ed. Chapman & Hall, New York and London. Franchi, L.L., Mandl, A.M., Zuckerman, S., 1962. The development of the ovary and the process of oogenesis (Chapter 1) In: Zuckerman, S., Mandl, A.M., Eckstein, P. (Eds.), The Ovary, 2 Volumes. Academic press, New York and London. Gayta´n, M., Sa´nchez, M.A., Morales, C., Bellido, C., Milla´n, Y., de las Mulas, J.M., et al., 2005. Cyclic changes of the ovarian surface epithelium in the rat. Reproduction. 129, 311 321. Hebel, R., Stromberg, M.W., 1976. Anatomy of the Laboratory Rat. The Williams & Wilkins Company, Baltimore, MD. Hunt, H.R., 1924. A Laboratory Manual of the Anatomy of the Rat. The Macmillan Company, New York. Long, J.A., Evans, H.M., 1922. The oestrous cycle in the rat and its associated phenomena. Mem. Univ. Calif. 6, 1 148. Mandl, A.M., 1951. The phases of the oestrous cycle in the adult white rat. J. Exp. Biol. 28, 576 584. McGee, E.A., Hsueh, A.J.W., 2000. Initial and cyclic recruitment of ovarian follicles. Endocr. Rev. 21 (2), 200 214. Paccola, C.C., Resende, C.G., Stumpp, T., Miraglia, S.M., Cipriano, I., 2013. The rat estrous cycle revisited: a quantitative and qualitative analysis. Anim. Reprod. 10 (4), 677 683.
229
Parkes, A.S., 1929. The Internal Secretions of the Ovary. Longmans, Green and Co, London. Porter, G., 1957. The Norway Rat (Chapter 32) In: Worden, A.N., LanePetter, W. (Eds.), The UFAW Handbook on the Care and Management of Laboratory Animals, second ed. Universities Federation for Animal Welfare, London, pp. 344 377. Rowett, H.G.Q., 1952. Dissection Guides: III The Rat (With Notes on the Mouse). John Murray, London. Tilly, J.L., Niikura, Y., Rueda, B.R., 2009. The current status of evidence for and against postnatal oogenesis in mammals: a case of ovarian optimism versus pessimism? Biol. Reprod. 80, 2 12. Van der Schoot, P., Emmen, J.M.A., 1996. Development, structure and function of the cranial suspensory ligaments of the mammalian gonads in a cross-species perspective; their possible role in effecting disturbed testicular function. Hum. Reprod. Update. 2 (5), 399 418. Waldeyer, W., 1870. Eierstock und Ei. Engelmann, Leipzig. Zuckerman, S., 1970. A story about mammalian egg-cells (Chapter 3, pp. 22 34) In: Zuckerman, S. (Ed.), Beyond the Ivory Tower. Weidenfeld and Nicolson, London.
FURTHER READING Parkes, A.S. Marshall’s Physiology of Reproduction. third ed. Longmans, Green and Co., London, New York, Toronto. Volume 1, part one: 1956; Volume 1, part two: 1960; Volume 2: 1952; Volume 3: 1966. (Irreplaceable as an account of the comparative anatomy of the reproductive system and of the physiology as understood at the time it was written. A Fourth Edition was produced: possibly incomplete). Zuckerman, S., Mandl, A.M., Eckstein, P., 1962. The Ovary. 2 Volumes. Academic press, New York and London (Invaluable for details of comparative anatomy).