Abstract
During preclinical investigations into the safety of drugs and chemicals, many are found to interfere with reproductive function in the female rat. This interference is commonly expressed as a change in normal morphology of the reproductive tract or a disturbance in the duration of particular phases of the estrous cycle. Such alterations can be recognized only if the pathologist has knowledge of the continuously changing histological appearance of the various components of the reproductive tract during the cycle and can accurately and consistently ascribe an individual tract to a particular phase of the cycle. Unfortunately, although comprehensive reports illustrating the normal appearance of the tract during the rat estrous cycle have been available over many years, they are generally somewhat ambiguous about distinct criteria for defining the end of one stage and the beginning of another. This detail is absolutely essential to achieve a consistent approach to staging the cycle. For the toxicologic pathologist, this report illustrates a pragmatic and practical approach to staging the estrous cycle in the rat based on personal experience and a review of the literature from the last century.
Introduction
During preclinical investigations into the safety of drugs and chemicals, many are found to interfere with the reproductive function of the female rat (Yuan and Foley 2002). This interference is commonly expressed as a change in normal morphology of the reproductive tract or a disturbance in the duration of particular phases of the estrous cycle. These alterations can be recognized only if the pathologist has knowledge of the continuously changing histological appearance of the various components of the reproductive tract during the cycle and can accurately and consistently ascribe an individual tract to a particular phase of the cycle. Indeed, functional alterations may be apparent only following such an assessment and may manifest themselves by a change in the number of animals in a particular phase of the cycle, a disturbance in the coordinated morphology of the components of the tract, or abnormal appearance of a particular region. Unfortunately, although comprehensive reports illustrating the normal appearance of the tract during the rat estrous cycle have been available over many years (Hubscher et al. 2005; Long and Evans 1922; Marshall 1960; Yuan and Foley 2002), they are generally somewhat ambiguous about distinct criteria for defining the end of one stage and the beginning of another. This detail is absolutely essential to achieve a consistent approach to staging the cycle. Indeed, some authors provide only a single description of regions of the tract from rats with a four-day estrous cycle, collected at a specific time on each day, as typical of a particular phase (Yuan and Foley 2002), which can lead to inconsistency when assessing progressive and cyclical changes. My aim here is to illustrate, for the toxicologic pathologist, a pragmatic and practical approach to staging the estrous cycle in the rat based on personal experience and literature references from the last century. The images presented (hematoxylin-and-eosin–stained sections processed by standard histological techniques) are examples from young adult undosed Wistar rats (Alderley Park strain) but are entirely consistent with, and illustrative of, other strains. I have also included descriptions of the appearance of the tract during aging. Some details of the associated hormonal changes are included in relation to the morphological changes, particularly for the aging rat, but they are necessarily cursory, as this is not the main purpose of the report. For a complete review of the hormonal correlates, see The Physiology of Reproduction, edited by Knobil and Neil (1994).
The Estrous Cycle
Estrus was used first by Heape (1900) as a Latin adaptation of the Greek word oistros, meaning gadfly, sting, or frenzy to describe the “special period of sexual desire of the female” (Freeman 1994). He also used: anestrus, nonbreeding season when reproductive organs are quiescent; proestrus, animal coming on heat; metestrus, in the absence of conception, when estrus changes in the reproductive tract subside; and diestrus, reproductive tract prepares for receipt of the ovum. Clearly, these “periods,” although they are accompanied by morphological changes, they are not described by them, although the morphological appearances of the reproductive organs of the rat have been well characterized under this general behavioral scheme, as noted in the introduction. Vaginal smears are also widely used for this purpose; their characteristics have been described in detail elsewhere (Hubscher et al. 2005; Long and Evans 1922; Maeda et al. 2000). However, they will also be discussed alongside the morphological features of the estrous stages under “Staging the Cycle” below in support of the proposed method.
The onset of puberty in the female rat results from a cascade of events following establishment of a pulsatile luteinizing hormone (LH) release after the fourth postnatal week (approximately thirty days of age) that leads to ovarian maturation (Andrews and Ojeda 1981). Before this time, the reproductive tract is inactive. This change in LH release is apparent eight to nine days before the first proestrus, and this period of change in mode of LH release is considered anestrus (Urbanski and Ojeda 1985). The first proestrus, estrus, and diestrus periods then follow (Advis et al. 1979; Ojeda et al. 1976).
Ovulation occurs in the young adult laboratory rat every four to five days throughout the year (Ojeda and Urbanski 1994). The normal length of the estrous cycle has been investigated over many decades. Long and Evans (1922) used approximately 2000 young adult rats derived from descendents of a cross between several white females and a wild grey caught in Berkeley, California, and fed on table scraps and other supplements, including raw liver. They found marked individual variations, with cycle lengths from three to thirty-eight days, but, excluding those of more than eight days, the average was 4.8 days, and this duration is reasonably consistent with findings from subsequent investigators (Astwood 1939; Blandau et al. 1941). Based on vaginal smears, the duration of the individual components of the estrous cycle for rats with a four- or five-day cycle are proestrus, twelve to fourteen hours; estrus, twenty-five to twenty-seven hours; metestrus, six to eight hours; and diestrus, fifty-five to fifty-seven hours (Astwood, 1939; Hartman 1944; Long and Evans 1922; Mandl 1951). However, as noted above, many authors refer to the day of the cycle, with each period having its own day, and those in a five-day cycle generally showing either an extra day of vaginal cornification (extra day of estrus) or an extra day of leukocyte infiltration (extra day of diestrus) (vom Sall et al. 1994).
Staging The Cycle
Complete longitudinal sections of the vagina and cervix, transverse sections of the mid portion of both uterine horns, and medial sections of both ovaries are the minimal requirement for an adequate evaluation.
Observation of the coordinated morphology of the vagina and uterus is key to consistent staging of the estrous cycle, and this will be the main focus of the descriptions provided here. The stage of the reproductive cycle cannot easily be determined from the appearance of the ovarian follicles in a species like the rat, which has a short estrous cycle. Follicles in virtually all stages of development are generally present within the ovaries at all phases of the cycle. However, the formation, progression, and regression of the corpora lutea are somewhat synchronized, and they can be used as an aid (Yuan and Foley 2002). It is of note that three or more generations of corpora lutea may be present in an individual ovary from the preceding ovulatory cycles, as each corpora lutea persists morphologically for twelve to fourteen days (Long and Evans 1922; Yuan and Foley 2002). Thus, the use of corpora luteal morphology as an accurate tool for estrous staging can be confusing. But the presence of particular morphologies of corpora lutea can be used to support staging, even though this is not definitive.
Table 1 summarizes the characteristics of the vaginal, uterine, and ovarian morphology during the four estrous stages seen in the normally cycling adult rat. These cycles are described below.
Diestrus
Figure 1 illustrates the diestrus stage.
Vagina
At the start of diestrus, the vaginal epithelium is at its lowest level of approximately three to seven cells thick consisting simply of the stratum germinativum. The stratum germinativum consists of stratum basale as a single layer of columnar epithelial cells and an outer stratum spinosum as multiple layers of polyhedral cells. There is a variable infiltration by leukocytes. A reduction in the infiltration of leukocytes and a notable epithelial cell proliferation occurs toward the end of the phase with thickening of the epithelium, but with no clear stratum granulosum. The formation of a stratum granulosum is the defining characteristic for the practical staging of the end of diestrus and the beginning of proestrus. The vaginal smear is consistent with this histological appearance, which is characterized by little mucus with some leukocytes, nucleated basophilic cells, and occasional vacuolated cells.
Uterus
At the start of diestrus, the uterus is small and inactive, and the horns lack a prominent vasculature and generally show a slit-like lumen. They are lined by a low cuboidal or columnar epithelium showing occasional degenerate cells. There are few mitoses at the start of the stage and the glands are particularly inactive, but there is some increase in activity during the progression of the phase. Also, toward the end of the phase, a slight edema of the stroma adjacent to the endometrial epithelium can be seen.
Ovaries
By diestrus, in the ovary, the newly formed corpora lutea from the previous ovulation have attained their maximal size, and this is the best ovarian marker for diestrus, although degenerate corpora lutea will also be present. Vacuoles are commonly present, particularly in the cells in the center of these large corpora lutea, indicative of active steroidogenesis, and early fibrous tissue formation may be seen in what was previously the central, fluid-filled cavity.
Proestrus
Proestrus is illustrated in Figure 2.
Vagina
Formation of the stratum granulosum over the stratum germinativum of the vaginal epithelium, consisting of flattened epithelial cells containing many keratohyalin granules, marks the start of proestrus. The vaginal epithelium shows mitotic figures throughout the stage, although they are less numerous at the end. Following the early formation of the stratum granulosum, there is a progressive development of the superficial mucoid layer (stratum mucification according to Yuan and Foley [2002], or rete mucosum according to Hubscher et al. [2005]), characterized by layers of cuboidal to ovoid cells with mucin-containing cytoplasmic vacuoles, and the formation of a stratum corneum of dense, cornified cells. There is little if any degeneration or desquamation during the early- or mid-proestrus period, and occasional infiltrates of leukocytes are seen. At the end of the stage, the epithelium is fully cornified and generally shows a superficial mucoid layer exhibiting some desquamation of mucoid cells. The vaginal smear is again consistent with this histological appearance, showing a disappearance of leukocytes and the presence of sheets of, or isolated, nucleated epithelial cells, which become progressively acidophilic with the appearance of cornified cells.
Uterus
During this stage, the uterine endometrial lining progresses to large cells, forming a tall cuboidal to columnar epithelium. There are frequent mitoses with only limited or no epithelial cell degeneration of the glands and lining epithelium (the appearance of notable endometrial epithelial cell degeneration marks the end of proestrus/start of estrus) and little inflammatory cell infiltration, although mitotic figures disappear at the end of the stage. The endometrial vasculature becomes more prominent, and the stroma can show some edema, with the lumen generally becoming markedly dilated toward the end of the stage.
Ovaries
Ovarian corpora lutea are often degenerate, with central fibrous tissue formation, and the cells commonly contain cytoplasmic vacuoles.
Estrus
Figure 3 illustrates estrus.
Vagina
In the vagina, there is a loss of mitotic figures and a progressive shedding of the superficial mucoid and cornified layers during estrus, with a reduction in the height of the epithelium, and cell debris is present in the lumen. There is also a variable and progressive leukocyte infiltration. The end of the stage is characterized by detachment of the cornified epithelium, although some may persist, particularly adjacent to the vaginal opening. So, if there is a predominance of attached cornified epithelium, then the stage is still “estrus.” Virtually complete detachment of the cornified epithelium of the vagina marks the end of estrus and the start of metestrus. The vaginal smear shows non-nucleated cornified cells, which by late estrus have diminished when leukocytes appear. Also, large basophilic epithelial cells are present in the smear.
Uterus
Changes in the endometrial epithelium define the start of estrus, with the appearance of cellular degeneration/necrosis in the glands first, followed by the lining epithelium, which becomes quite marked. It is accompanied by a loss of mitotic activity and leukocyte infiltration. Luminal dilatation may persist into late estrus, although this is generally not the case. Some endometrial epithelial mitotic activity returns by the end of estrus.
Ovaries
Degenerate ovarian corpora lutea are often present at estrus, but others (the newly formed corpora lutea) are small, with cells showing a basophilic cytoplasm and occasionally a central fluid-filled cavity retained from the follicular stage. Central fibrous tissue is generally not present.
Metestrus
Metestrus is illustrated in Figure 4.
Vagina
The start of metestrus is marked by the mid region of the vagina showing a complete detachment of the cornified epithelium, generally with residual squames present in the lumen. As noted above, some cornified epithelium may persist, particularly adjacent to the vaginal opening. There is a continued desquamation of epithelial cells throughout the stage, with a progressive loss of the stratum granulosum and upper germinativum (stratum spinosum). There is an accompanying variable leukocyte infiltration. As noted under diestrus, the end of metestrus/start of diestrus is marked by the epithelium reaching its lowest level. The smear during this stage shows leukocytes, a few cornified cells, and basophilic cells.
Uterus
During metestrus, the uterine endometrial epithelium shows continued vacuolar degeneration, but also a marked return of mitotic activity, so both are seen together. There is also a variable leukocyte infiltration
Ovaries
Ovarian corpora lutea may still contain a fluid-filled central cavity generally devoid of fibrous tissue, and the cytoplasm of the new corpora lutea are somewhat less basophilic than at estrus but smaller than that seen at diestrus.
The Cycle in Aging Rats
During the midlife period in rats, the estrous cycle length increases, and some histological inconsistency is occasionally seen between the various components of the tract. Progesterone plays an important role in influencing cycle length in rodents (Nequin et al. 1979), and progesterone and estrogen act synergistically (Morali and Beyer 1979). The estrogen/progesterone ratio is elevated for prolonged periods before ovulation in most middle-aged rats, and this age-related deficit in progesterone has a significant responsibility for this increase in cycle length (vom Saal et al. 1994). At any time from six to eighteen months, but seldom later, following lengthening of the cycle or interspersed long cycles, a constant state of vaginal cornification or “persistent estrus” can be observed (vom Saal et al. 1994). This may be followed by states in which cycles are ten to fourteen days long, suggestive of a prolonged luteal phase, which is termed “repetitive pseudopregnancy” (Nelson and Felicio 1985). The final stage of reproductive senescence is “persistent anestrus” (vom Saal et al. 1994).
Persistent Estrus
Persistent estrus is illustrated in Figure 5.
Subsequent to the lengthening of the estrous cycle, most aging females between six and eighteen months of age exhibit periods of persistent estrus, with constant sexual receptivity (Felicio et al. 1984). This state results from estradiol levels being maintained at a tonic level (Lu et al. 1979; Nelson et al. 1981), reflecting the presence of numbers of ovarian follicular cysts (Gosden, Laing, Flurkey et al. 1983) and a tall columnar endometrial epithelium. The tonic estrogen secretion stimulates cornification of the vaginal epithelium, so this state is also referred to as persistent vaginal cornification (vom Saal et al. 1994). Low levels of progesterone during this period (Miller and Riegle 1980; Nelson et al. 1981) are considered responsible for the maintenance of this state (vom Saal et al. 1994) and are associated with an absence of corpora lutea (Gosden, Laing, Flurkey et al. 1983).
Repetitive Pseudopregnancy
Repetitive pseudopregnancy is illustrated in Figure 5.
In young adult female rats, whether sterile or fertile, for the first nine or ten days after mating, prolactin secreted by the pituitary maintains the corpora lutea. The placenta of a successfully mated female then takes over (Smith 1980; Smith et al. 1975). If mating is unsuccessful, the corpora lutea degenerate by Day 12 or 13.
Therefore, the repetitive states of pseudopregnancy that can occur following persistent estrus in aging rats are typically ten to fourteen days in duration (vom Saal et al. 1994). This period is characterized by maintenance of corpora lutea beyond the normal two-day life span and is associated with high progesterone levels (vom Saal et al. 1994). It is considered that these changes, following an elevated estradiol/progesterone ratio during persistent estrus, result from reduced estradiol and elevated prolactin, often as a result of a pituitary tumor (Everett 1980). The raised prolactin levels maintain persistence of the corpora lutea. The vagina during this period shows a low stratum germinativum, but variable overlying mucification. The uterus commonly exhibits a folded endometrial epithelium.
Persistent Anestrus
Figure 6 illustrates persistent anestrus.
Old rats eventually reach a final stage of reproductive senescence. This is termed persistent anestrus, or persistent diestrus. After the cessation of ovulation and entry into persistent estrus, there is still some maturation of follicles, followed by atresia without ovulation, culminating in complete follicular depletion (Gosden, Laing, Felicio at al. 1983; Gosden, Laing, Flurkey et al. 1983). Morphological features of the reproductive tract are characterized by a low, somewhat mucified vaginal epithelium and a low inactive endometrial epithelium. The loss of follicles results in estradiol levels similar to those seen in ovariectomized rats (vom Saal et al. 1994). There are low progesterone levels, reflecting the absence of corpora lutea (Lu et al. 1979), and high prolactin levels, generally from pituitary tumors, which have an inhibitory effect on LH (vom Saal et al. 1994).