The Guinea Pig Estrous Cycle: Correlation of Vaginal Impedance Measurements with Vaginal Cytologic Findings

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1 Laboratory Animal Science Copyright 1997 by the American Association for Laboratory Animal Science Vol 47, No 6 December 1997 The Guinea Pig Estrous Cycle: Correlation of Vaginal Impedance Measurements with Vaginal Cytologic Findings Kenneth G. Lilley, Ronald J. Epping, and Louise M. Hafner Abstract Immunologic responses elicited after vaginal immunization of laboratory mammals are affected by the stage of the animal s estrous cycle at which antigen is delivered. The study reported here documents vaginal impedance as an improved means of determining accurately precise stages of the estrous cycle of guinea pigs. Impedance changes were correlated with cytologic changes observed in vaginal smears taken from the animals. The start of the impedance peak corresponded with proestrus, the rising side of the peak with estrus, and the apex of the peak with metestrus; diestrus was seen approximately 3 days after the peak apex. These results contrast with those of previous studies. Various dosage regimens of estradiol (1 to 1,000 g/animal over 1 to 6 days) were used in guinea pigs in an attempt to invoke an extended (>6 h) estrogeninduced mucosa. Parenteral administration of 1,000 g of estradiol to guinea pigs daily for 6 days induced a 2- to 3-day estrus stage in these animals. Immunization strategies for diseases affecting the systemic organs of humans have been available for many years and have reduced considerably the incidence and mortality of these diseases. There has, however, been a remarkable lack of success in vaccination strategies used to control mucosal diseases (1), such as those caused by Salmonella and Vibrio spp., and human immunodeficiency virus. Chlamydia trachomatis is a particularly good example because, despite the fact that this obligate intracellular bacterium is a leading cause of sexually transmitted disease in humans, affecting millions of people throughout the world (2), there is no effective vaccine for this mucosal pathogen (3). Local mucosal immunization of laboratory animals with antigen has been shown to induce an increased mucosal immune response at the site of exposure, compared with the response elicited after immunization at distant mucosal sites (4, 5). Because chlamydiae invade the body via mucosal surfaces, such as the female reproductive tract (FRT), an important requirement for an effective vaccine against these bacteria is that it must stimulate the mucosal immune system (6). Consequently, the body site used for immunization of individuals with antigen preparations is an important consideration in attempts to raise immunity to chlamydial and other infections. The most common animal model used to investigate mucosal immune mechanisms involved in chlamydial infections of the genital tract is the guinea pig, in which Centre for Molecular Biotechnology, School of Life Sciences, Queensland University of Technology, GPO Box 2434 Brisbane Q 4001, Australia infection has been shown to closely parallel genital infections of humans with respect to pathogenesis and pathologic and immunologic findings of disease (7, 8). A current focus of world-wide mucosal vaccinology involves endocrinology of the estrous cycle, especially because concentrations of the female sex hormones (particularly estrogen) also have been shown to have pronounced effects on the intensity and duration of chlamydial infection in guinea pigs (9, 10). The guinea pig has an estrous cycle of 15 to 17 days duration that consists of four sequential stages: estrus, metestrus, diestrus, and proestrus (11). The stage of the estrous cycle affects the extent of antigen uptake in the FRT of mice (12) and the presence of antigen-presenting cells (lowest during estrus) in rats (13). Estrus is estrogen dominated, diestrus is progesterone dominated (14), and concentrations of sex hormones have been reported to affect the pathogenesis of infection (15). The stage of the estrous cycle also is emerging as an important consideration when planning the timing of antigen delivery for mucosal vaccination; however, a convenient method to identify accurately and monitor the stages of the guinea pig estrous cycle is unavailable. Vaginal cytologic examination commonly is used to monitor the guinea pig estrous cycle, but it is time consuming and requires experience with the technique. The guinea pig has a vaginal closure membrane that covers the vaginal orifice throughout the estrous cycle and that degenerates spontaneously at estrus (16). The absence of a vaginal closure membrane in guinea pigs also may be used to indicate estrus (17), although correlation is not 100% (18). It has been suggested that vaginal impedance (rather than cytologic examination of vaginal smears) may better correlate with stages of the estrous cycle (19). 632

2 Identification of Estrous Cycle Stages Using Vaginal Impedance Table 1. Induction of estrus by estradiol administration. Twenty-nine female guinea pigs each were given 0 to 1,000 g of estradiol over a 6-day period in an attempt to induce a prolonged estrus mucosa Group N Induction Dosage Result 1 5 Multiple 1 g/animal per day for 6 days Delayed subsequent cycle by 2 days 2 5 Multiple 10 g/animal per day for 6 days Enhanced epithelium maturation 3 5 Multiple 100 g/animal per day for 6 days Irregular cycle, enhanced epithelium maturation 4 5 Single 100 g/animal Normal cycle, enhanced epithelium maturation 5 4 Multiple 1,000 g/animal per day for 6 days Induced estrus 6 5 Multiple 100 l of arachis oil/animal per day for 6 days No effect N = number of animals in each experimental group. We investigated the use of vaginal impedance measurements to monitor the guinea pig estrous cycle. We also in vestigated the correlation between vaginal cytologic features and vaginal impedance measurements throughout the cycle. The impedance meter also was used in conjunction with estradiol administration to the a nimals to investigate potential induction of prolonged estrus in these animals to facilitate vaginal immunization. Materials and Methods Animals: Virgin female random outbred English shorthaired (ROES) strain guinea pigs were obtained from Monash Animal Services (Melbourne, Victoria, Australia). The animals were sexually mature (>60 days old), and each weighed 500 to 900 g. Microchips (Trovan, Victoria) were injected subcutaneously (dorsoscapular) into the animals, and a scanner (Trovan LID500; Central Animal Register, Victoria) was used for individual identification. Animals were housed in an environmentally controlled room (21 1 C, 12/12-h light/dark cycle) at the University of Queensland Medical School Animal Facility. Standard guinea pig pellets (Norco Cooperative Ltd., Lismore, New South Wales, Australia) and tap water supplemented with ascorbic acid (4 mg/20 L; Melrose Laboratories, Victoria) were available ad libitum. This study was done in compliance with all relevant regulations regarding safety, ethics, and use of animals, and all procedures were approved by the Queensland University of Technology Biomedical Ethics Committee (BEC no. 662). In the case of the guinea pigs, procedures also were approved by the University of Queensland Animal Experimentation Ethics Committee (AEEC approval no. QUT/060/ 95/NHMRC). Estrous cycle studies. Vaginal cytology: Vaginal smears were prepared from specimens obtained daily at 1600 h from 29 female guinea pigs over 135 days, using the following procedure. To obtain a vaginal swab specimen, the anogenital area of a restrained guinea pig was cleansed, using cotton wool moistened with sterile phosphate-buffered saline solution (PBSS). If the vaginal membrane was present, it was gently perforated with a sterile cotton-tipped aluminum swab (Disposable Products, South Australia). Smears were collected from the proximal third of the vaginal epithelium by gently inserting a moist, sterile swab 2 cm into the vagina and twice rotating it vigorously against the vaginal wall. The swab immediately was rolled onto a glass microscope slide, and the cells were fixed by spraying with Cytospray (SEA Trading Pty Ltd, Queensland, Australia). The smears were stained (20) and permanently mounted, using Depex (Labtek, Queensland). Vaginal smears were evaluated, using standard cytologic criteria for diagnosis of stage of the estrous cycle (21), and were examined, using an Olympus CH2 microscope at magnifications of 100x and 400x. Cell types and their distribution were assessed on a 1+ to 4+ scale, and stage of the estrous cycle was determined (11, 16). Vaginal smears were prepared from specimens collected from 29 guinea pigs over 135 days and were stained by use of the Papanicolaou method to identify the cell types and their relative frequencies throughout the estrous cycle. The stained slides were examined in coded experiments to avoid bias, and these data were used to differentiate stages of the estrous cycle in these animals. Vaginal impedance measurements: Electric impedance of the vaginal mucous membrane was measured daily at 1600 h in 43 female guinea pigs over 135 days as described by Bartos and Sedlacek (19). Briefly, a probe containing two electrodes was inserted 2 cm into the vagina, and the impedance was measured by the attached impedance meter. The meter was a transistor indicator with a 2-kHz oscillator that was based on a meter constructed by Petran (22). The probe was cleaned in 70% alcohol before each guinea pig measurement. Daily vaginal impedance measurements were obtained from the 43 guinea pigs over a period of 135 days to ascertain whether these varied throughout the estrous cycle and whether they correlated with cytologic changes seen in the animals. To further delineate cytologic changes and vaginal impedance changes where impedance peaked, impedance measurements were obtained and smears were prepared every 3 h for 48 h. Induction of estrus by estradiol administration: Estradiol benzoate (Intervet, New South Wales) was administered subcutaneously (s.c.) to 29 female guinea pigs, using 100- l volumes of various dosages (1 to 1,000 g, Table 1); arachis oil diluent for the estradiol benzoate (Health Mindery, New South Wales) was given to control animals. Six groups of five guinea pigs (groups 1 4, and 6) and one group of four guinea pigs (group 5) were used. Animals were given estradiol benzoate at 1700 h daily for 6 days, except for those of group 4, which were given 100 g of estradiol benzoate once only, and those of group 6 (controls), which were given 100 l of arachis oil daily for 6 days. Results Correlation between vaginal cytologic findings and impedance measurements taken during the estrous cycles of guinea pigs. Vaginal cytologic examination: Vaginal cytologic features at estrus consisted 633

3 Vol 47, No 6 Laboratory Animal Science December 1997 Figure 1. Representative photomicrographs of Papanicolaou-stained vaginal smears from guinea pigs illustrating the relative cellular composition of the four stages of the estrous cycle. Estrus (upper left); metestrus (upper right); proestrus (lower left); diestrus (lower right). SSC = superficial squamous cell, may be nucleated (n) or non-nucleated (nn); ISC = intermediate squamous cell; PB = parabasal cell; PMN = polymorphonuclear leukocyte. Central arrows indicate progression of the estrous cycle. Magnification = x200. predominantly of non-nucleated superficial squamous cells (SSC[nn]), whereas those seen during metestrus were a mixture of cell types, including SSC(nn), nucleated superficial squamous cells (SSC[n]), and parabasal cells (PB). Metestrus was characterized by a marked influx of polymorphonuclear neutrophils (PMN, Figure 1). Cells seen during diestrus consisted mainly of PBs and PMNs; generally SSCs were lacking. Proestrus was confirmed by presence of intermediate squamous cells (ISC); SSCs (nucleated and non-nucleated) and PBs also were seen during proestrus, and PMN numbers were reduced. There was overlap in cell types observed between each of the stages of the cycle; however, differentiation of each stage was facilitated by adherence to a quantitative assessment of the cell types and their frequencies for each stage, using a scale of 1+ to 4+ (Table 2). Investigation of vaginal impedance: A cyclic increase in electric impedance was evident in 98% of the guinea pigs 634

4 Identification of Estrous Cycle Stages Using Vaginal Impedance Table 2. Morphologic cell types and their relative frequencies seen in vaginal smears prepared from specimens collected from guinea pigs at the four stages of the estrous cycle Cell types and relative frequencies Stage of Duration Superficial Intermediate Parabasal Polymorphonuclear estrous cycle of stage squamous squamous cells leucocytes cells cells Estrus 6 11 h Metestrus 2 4 day 2+ to 4+ nn to to 4+ Diestrus 8 10 day to to 3+ Proestrus 2 4 day 1+ to 2+ nn 2+ to to to 2+ n Vaginal smears were stained, using the Papanicolaou method; cell types in the smears were identified and their relative frequencies were determined in relation to the other cell types and their distribution over the entire smear. Relative frequencies of cell types ranged from 1+ (0 to 10%) to 4+ (80 to 100%). Superficial squamous cells (SSCs) were further classified into nucleated (n) and non-nucleated (nn) SSCs. The data were compiled from the analysis of 3,915 smears. in this study; data from a typical guinea pig response are shown in Figure 2. One guinea pig appeared to lack an estrous cycle; it remained in cytologic diestrus, and an impedance peak was not evident. For all 42 guinea pigs exhibiting cyclic vaginal impedance, the periodicity of the impedance peak corresponded with an estrous cycle duration of 15 to 17 days (mean 2 SD, days). Mean amplitude of the vaginal impedance peak was ( 2 SD), and mean impedance for the remainder of the cycle (nonpeak) was ( 2 SD); the difference was statistically significant (P < 0.01, t test). Vaginal impedance correlated with cytologic changes in the estrous cycle of all guinea pigs monitored. A typical guinea pig response is shown in Figure 3. Proestrus was cytologically evident 3 days prior to the start of the peak and for 2.5 days into the rising side of the peak. Estrus was evident for approximately 12 h as impedance was increasing. Metestrus was observed at the highest point of the impedance peak and for 2.5 days after return of impedance to nonpeak values. Diestrus was cytologically evident after metestrus, commencing 2.5 days after the impedance peak. Induction of estrus in guinea pigs by estradiol administration: Various dosages of estradiol were administered to five groups of guinea pigs (five animals in groups 1, 2, 3, and 4; four animals in group 5) in an attempt to evoke a prolonged (2- to 3-day) estrogen-induced mucosa instead of the typical 6 to 11 h, thereby facilitating immunization at that stage of the cycle (Table 1). The stage of the guinea pig estrous cycle was monitored by vaginal impedance measurements and by examination of Papanicolaou-stained vaginal smears prepared from specimens taken daily at 1600 h. Data were screened for signs of estrus induction, such as increase in vaginal impedance, enhanced maturation of the vaginal epithelium (i.e., increased presence of cell types usually present at later stages of the estrous cycle), or absence of the vaginal closure membrane. Changes in the estrous cycle of animals in group 1 (1 g of estradiol/animal daily for 6 days) included delayed commencement of the next cycle by 2 days (3/3 animals) and enhanced maturation of the vaginal epithelium for 5 days after the next stage of the estrous cycle (5/5 animals). For 5 of 5 animals in group 2 (10 g of estradiol) the subsequent cycle was not delayed, but maturation of the vaginal epithelium was enhanced from the onset of estrus and continued in an irregular manner to the second cycle after es- tradiol administration. In group-3 (100 g of estradiol) guinea pigs, commencement of the next cycle was irregular (i.e., 1 to 2 days in 3/5 animals) and started 5 days earlier in 2/5 animals. All animals in this group had enhanced vaginal epithelium maturation for 3 to 5 days after estrus. In group-4 (single dose of 100 g of estradiol) guinea pigs, the following two cycles were of normal duration, but enhanced vaginal epithelium maturation was evident for 1 to 2 days after estrus. In group-5 (1,000 g of estradiol) guinea pigs, enhanced epithelium maturation was evident from days 13 to 14 after the first estradiol injection, culminating in extended estrus, lasting 2 to 3 days from days 21 to 23. Thus, unlike the 1-, 10-, and 100- g doses of estradiol per animal (which caused enhanced vaginal epithelium maturation, but not complete estrus induction), the 1,000- g dose given daily to each animal for 6 days resulted in induced estrus. Numbers of PMNs observed at estrus usually were low (often zero), whereas numbers of PMNs observed during estradiol-induced estrus were scored as 1+ to 2+. For all groups of animals, enhanced maturation of the vaginal epithelium preceded the increase in vaginal impedance by 1 to 2 days. In animals of the 1,000 g/animal dosage group, the vaginal closure membrane was absent (a sign of estrus) from the start of induced estrus and failed to re-form until 7 days after the commencement of induced estrus (c.f., re-forming of the membrane the day after noninduced or normal estrus). In animals of the lower-dosage groups, there was no consistent correlation between induced vaginal epithelium maturation and absence of the vaginal closure membrane. Administration of arachis oil to the control animals in group 6 (Table 1) did not affect the estrous cycle. Discussion Cell types (and their frequencies) in vaginal smears prepared from guinea pigs at various stages of the estrous cycle were in accordance with published findings (11, 16), except for the terminology used to describe a type of SSCs found in the stained smears. Previous studies referred to a cell type termed SCC(n), whereas in the study reported here, further differentiation of this type of cell was made. Using cytologic criteria, principally nuclear morphology, the SSC(n) was differentiated from another cell type, the intermediate squamous cell (ISC) (23). This is an important distinction, because proestrus is characterized cytologically by the presence of 2+ to 3+ ISCs. 635

5 Vol 47, No 6 Laboratory Animal Science December 1997 Figure 2. Changes in vaginal impedance in one guinea pig over 6 estrous cycles. Vaginal impedance measurements were obtained from guinea pigs daily at 1600 h. The cyclic impedance relationship for this guinea pig also was evident for 42 of 43 animals in the study. Duration of the estrous cycle was determined cytologically; however, differentiating the stages of the cycle was difficult because the stages merge together. Because estrus generally occurs between 1800 and 0600 h (19), it often was missed when the smears were prepared from specimens taken at 1800 h. By strictly following descriptions of stages of the estrous cycle, as described in Table 2, it was possible to identify the different stages by cytologic examination; however, the divisions between each cycle stage were not easy to define. To predict confidently the onset of an estrous stage, serial smears had to be prepared from specimens collected daily over the duration of at least one complete estrous cycle (16 days). Assessment of vaginal cytologic features in smears is time consuming and requires cytologic knowledge and experience. Vaginal impedance measurements, however, take only seconds to obtain and are less amenable to subjective interpretation. The cyclic increase in impedance seen in the guinea pigs of this study was similar to that seen previously (19); however, the magnitude of the readings in this study differed. Bartos and Sedlacek (19) used a vaginal probe with ring electrodes of separation (3 mm) different from those used in this study (1 mm); this may account for the approximately threefold higher values reported in their study. The 16 ( 1)-day duration of the estrous cycle of the guinea pigs of this study is in full accordance with the 16.3 days of one report (24) and the 16.6 days of another report (19). Bartos and Sedlacek (19) made certain assumptions about impedance values and estrous stages in guinea pigs on the basis of previous work by Bartos (25), using rats. The study reported here is the first to investigate the relationship between cytologic and vaginal impedance changes in the guinea pig over the estrous cycle, and our findings disagree with the assumptions of Bartos and Sedlacek (19). Those authors assumed that the highest impedance values corresponded to proestrus, whereas the results of this study clearly indicate that proestrus occurs prior to and during the vaginal impedance peak and that the highest impedance value actually corresponds to late estrus and metestrus (Figure 3). Figure 3. Correlation of vaginal impedance with stages of the guinea pig estrous cycle, as determined by vaginal cytologic examination. Vaginal impedance measurements and vaginal smears were taken daily at 1600 h for 135 days. To further investigate the peak region, smears were examined and impedance measurements were obtained from 5 guinea pigs every 3 h for 48 h in the region of the impedance peak. Cytologic changes consistent with the estrous cycle stages were observed at the intervals noted: D = diestrus, P = proestrus, E = estrus, and M = metestrus. Proestrus can be detected by noting the start of the impedance peak (i.e., 400 ). Estrus was difficult to detect because of its short duration of 6 to 11 h (26), but metestrus could be identified by the highest impedance value of the cycle. Because metestrus lasts 2 to 4 days (26), diestrus easily was identified as occurring 5 days after peak impedance. To monitor and distinguish all stages of the guinea pig estrous cycle, vaginal impedance changes, like vaginal cytologic changes, need to be measured daily and followed for at least one complete estrous cycle. Impedance measurements are easy to perform, faster to assess, and more effective for identifying stages of the estrous cycle for immunization than is examination of vaginal smears; therefore, impedance measurement is a valuable method to use in place of examination of vaginal smears. Nevertheless, due to the comparatively short duration (6 to 11 h) of the estrus stage of the cycle, a confirmatory vaginal smear also should be prepared, stained, and examined to confirm the estrous stage. Apart from an early report (27) containing irregular results, studies involving administration of estradiol to guinea pigs for the purposes of investigating hormonal effects on disease processes have not assessed effects on the estrous cycle after administration of the hormone. Administration of estradiol (1,000 g/animal per day for 6 days) to a group of guinea pigs (A. McCracken, unpublished results, this laboratory) resulted in enhanced maturation of the vaginal epithelium and an induced estrus in these animals that parallelled results of this study. The increase in PMNs observed during the induced estrus may have been due to a physiologic response to the prolonged absence of the vaginal closure membrane. Lower dosage regimens of estradiol tested in this study affected the duration and maturation of the estrous cycle; however, they did not induce estrus for an extended period. Administration of 1,000 g of estradiol/animal over 6 days was successful in causing an induced estrus for an extended period of 2 to 3 days. 636

6 Identification of Estrous Cycle Stages Using Vaginal Impedance From results of this study, it now is possible to administer estradiol at a dosage known to induce estrus for a period during which guinea pigs can be immunized with chlamydial or other antigen for the purpose of eliciting a local immune response. Alternatively, serial vaginal impedance measurements may be taken in nonestradiol-induced animals, and when impedance starts to increase, true estrus can be confirmed by vaginal cytologic examination, and the vaccine can be delivered. Further research is required to ascertain whether this induced estrus differs from true estrus in type and distribution of immunologically active cells, such as antigen-presenting cells, because this may have a profound effect on the efficacy of vaccine preparations delivered intravaginally. Acknowledgements The authors thank R. Dickson, Laboratory Manager of the University of Queensland Medical School Animal Facility, and his technical staff, Ms. S. Deitrich and Ms. C. Smith, for assistance with the maintenance and care of the animals. Mr. Dickson also is acknowledged for provision of the impedance meter used in these studies. This research was supported financially by a grant from the National Health & Medical Research Council of Australia to P. Timms, L. Hafner, R. Epping, and M. O Brien. References 1. Husband, A. J Novel vaccination strategies for the control of mucosal infection. Vaccine 11(2): Moulder, J. W., T. P. Hatch, C. C. Kuo, et al The Chlamydiae, p In N. R. Krieg (ed.), Bergey s manual of systemic bacteriology, vol 1. Williams & Wilkins, Baltimore. 3. Raulston, J. E Chlamydial envelope components and pathogen-host cell interactions. Mol. Microbiol. 15: Ogra, P. L., and S. S. Ogra Local antibody response to poliovaccine in the human female genital tract. J. Immunol. 110: Menge, A. C., S. M. 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