Cytologic hormonal and ultrasonographic correlates of the menstrual cycle of the New World monkey Cebus apella.код для вставкиСкачать
American Journal of Primatology 66:233–244 (2005) RESEARCH ARTICLE Cytologic, Hormonal, and Ultrasonographic Correlates of the Menstrual Cycle of the New World Monkey Cebus apella R.E. ORTIZ1, A.C. ORTIZ1, G. GAJARDO1, A.J. ZEPEDA2, V.H. PARRAGUEZ3, M.E. ORTIZ1n, and H.B. CROXATTO1,4 1 Unit of Reproductive Biology and Development, Faculty of Biological Sciences, Catholic University of Chile, Santiago, Chile 2 Instituto Chileno de Medicina Reproductiva (ICMER), Santiago, Chile 3 Faculty of Veterinary and Animal Sciences, University of Chile, Santiago, Chile 4 Millenium Institute for Fundamental and Applied Biology, Santiago, Chile Few reports on the reproductive physiology of Cebus apella have been published. In this study we characterized menstrual cycle events by means of vaginal cytology, ultrasonography (US), and hormonal measurements in serum during three consecutive cycles in 10 females, and assessed the probability that ovulation would occur in the same ovary in consecutive cycles in 18 females. The lengths and phases of the cycles were determined according to vaginal cytology. Taking the first day of endometrial bleeding as the first day of the cycle, the mean cycle length 7 SEM was 19.570.4 days, with follicular and luteal phases lasting 8.270.2 and 11.370.4 days, respectively. The follicular phase included menstruation and the periovulatory period, which was characterized by the presence of a large number of superficial eosinophilic cells in the vaginal smear. The myometrium, endometrium, and ovaries were clearly distinguished on US examination. During each menstrual cycle a single follicle was recruited at random from either ovary. The follicle grew from 3 mm to a maximum diameter of 8–9 mm over the course of 8 days, in association with increasing estradiol (E2) serum levels (from 489741 to 1600792 pmol/L). At ovulation, the mean diameter of the dominant follicle usually decreased by >20%, 1 day after the maximum E2 level was reached. Ovulation was associated with an abrupt fall in E2, a decreased number of eosinophilic cells, the presence of leukocytes and intermediate Contract grant sponsor: UNDP/UNFPA/WHO/World Bank Special Programme of Research, Development and Research Training in Human Reproduction; Contract grant number: 98/LABENDO/RMG-2; Contract grant sponsor: Rockefeller Foundation; Contract grant number: RF 98024#98. n Correspondence to: Marı́a Elena Ortı́z, Unit of Reproductive Biology and Development, Faculty of Biological Sciences, Catholic University of Chile, Casilla 114-D., Santiago, Chile. E-mail: email@example.com Received 27 August 2004; revised 15 November 2004; revision accepted 15 January 2005 DOI 10.1002/ajp.20141 Published online in Wiley InterScience (www.interscience.wiley.com). r 2005 Wiley-Liss, Inc. 234 / Ortiz et al. cells in the vaginal smear, and a progressive increase in progesterone (P) levels that reached a maximum of 892765 nmol/L on days 3–6 of the luteal phase. The menstrual cycle of Cebus apella differs in several temporal and quantitative aspects from that in humans and Old World primates, but it exhibits the same correlations between ovarian endocrine and morphologic parameters. Am. J. Primatol. 66:233–244, 2005. r 2005 Wiley-Liss, Inc. Key words: ovarian cycle; ultrasonography; New World primate; Cebus apella; ovulation side; Markov chain INTRODUCTION Our increasing knowledge and understanding of the reproductive biology of nonhuman primates continues to expand our awareness of the diversity of species and processes. In some cases it may be useful to deal with animals with poor or excessive reproduction, and it is essential to characterize their usefulness as animal models for health problems that affect human beings. The New World monkey Cebus apella (capuchin monkey) is being used successfully to advance our knowledge about the reproductive processes of primates. Most of the available information on the physiology of the menstrual cycle in this species is derived from reports by Nagle et al. [1979, 1980] and Nagle and Denari . These authors described the vaginal cytology and temporal relationships between the plasma profile of a luteinizing hormone (LH)-like activity, estradiol (E2) and progesterone (P), and ovulation determined by radioimmunoassay (RIA) and serial laparoscopy, respectively. However, other processes related to the menstrual cycle, such as fertilization, ovum transport, early development, and implantation, have not yet been described. Ultrasound (US) techniques allow repeated noninvasive examinations of internal organs, and have been used to assess follicular development, ovulation, luteal status, and the endometrium in various subjects, including women [Gormaz et al., 1992; Katayama, 1990; Kerin et al., 1981; Randall et al., 1989; Zegers-Hochschild, 1988], domestic animals [Pierson & Ginther, 1984, 1985], and rhesus monkeys [Morgan et al., 1987]. In this study we examined the cytologic, hormonal, and US correlates of the menstrual cycle of cebus monkeys in captivity, and the side of ovulation in consecutive cycles. MATERIALS AND METHODS Subjects For this study we used 22 sexually mature female monkeys (Cebus apella) that had lived for at least 4 years in the indoor colony of the Catholic University of Chile. Six animals came from the Centro Argentino de Primates (CAPRIM); three came from the Instituto Veterinario de Investigaciones Tropicales y de Altura (IVITA), Iquitos, Perú; 12 came from the Sao Pablo Zoo, Brazil; and one was born in our colony. The animals were 6–10 years old according to published anthropometric values for Cebus apella [Moreno Azorero & Rosner, 1989]. They weighed 2–3 kg, and all had experienced at least two consecutive menstrual cycles of normal duration (17–22 days) within the last 2 months. Each female was caged individually under controlled conditions (24–271C, 70% relative humidity, and a 14L:10D photoperiod). Tap water was available ad libitum, and fresh and dried Cebus apella Ovarian Cycle / 235 fruits, pelleted chow, biscuits, and a cake containing milk, minerals, eggs, honey, and corn were provided daily. Before each female was entered into the study she was submitted to daily vaginal smears until vaginal bleeding was detected. The first day of bleeding was taken as the first day of the cycle. Animal care and experimental procedures were done according to the guidelines of the Institutional Ethics Committee of the Catholic University of Chile. Experimental Design Experiment 1. We monitored 10 females throughout three consecutive cycles in order to examine relationships between follicular growth, ovulation, endometrial and myometrial thickness, vaginal cytology, and hormonal parameters. The first and third cycles (n=20) were monitored by vaginal smears, blood sampling, and US examinations, and the second cycle (n=10) was monitored only by vaginal smears and US. We obtained vaginal smears daily to identify the phases of the cycle and to determine its length (according to Nagle and Denari ). Blood sampling and US examinations were done every other day during the follicular phase, daily during the periovulatory period (see below), and every 2 days during the luteal phase to measure E2 and P, and to assess follicular growth, ovulation, and endometrial and myometrial thickness. Experiment 2. We monitored 18 females throughout six consecutive cycles to determine the side of the ovulatory follicle. Four of these females were monitored again for the same period 1 year later. These cycles were monitored by daily vaginal smears and one or two US examinations of the leading follicle during the periovulatory period. Hormone Assays We obtained blood samples (1.5 ml) by puncturing the saphenous vein of the animals while they were under ketamine hydrochloride 10 mg/kg i.m. anesthesia (Ketostops; Drag Pharma Invetec, Santiago, Chile) supplemented with atropine sulphate 0.04 mg/kg (Reg. I.S.P. F-1221-98; Laboratorio Sanderson S.A., Santiago, Chile). Blood samples were obtained every other day throughout the cycle, and daily during the periovulatory period and early luteal phase. After centrifugation, the serum was separated and stored at 201C until it was assayed for hormones. E2 and P were measured by a previously validated RIA for Cebus apella [Recabarren et al., 1998] and with the reagents supplied by the WHO Matched Reagent Program. Assays were done in duplicate from reconstituted aliquots of diethyl ether extracts. The antibody for E2 was raised against estradiol-6-CMOBSA, and that for P was a monoclonal antibody raised against progesterone-3CMO-BSA. The lower limits of sensitivity for E2 and P were 117 pmol/L and 35 nmol/L, respectively. The inter- and intra-assay coefficients of variation were 15% and 6% for E2, and 13% and 7% for P. US Examination We scanned the animals transabdominally while they were under ketamineatropine anesthesia, using a real-time B-mode US scanner (ALOKA SSD-1100 236 / Ortiz et al. ‘‘FLEXUS’’; Aloka Co., Ltd., Tokyo, Japan) fitted with a 7.5 MHz high-density electronic convex probe. Structures as small as 1 mm can be resolved with this equipment. The uterus and endometrium were measured in the sagittal and transverse planes. We determined the thickness of the uterus and endometrium by calculating the mean of one lateral and two dorsoventral measurements. Using the uterus as a landmark, we moved the transducer slowly in a transverse plane to locate the ovaries. These appear as ovoid structures with hyperechoic borders. The position of the ovarian vessels within the ligamentum suspensorium ovarii was used as a reference to identify the ovaries. Follicles Z3 mm were measured with omnidirectional calipers. The size of each ovary, follicle, and corpus luteum (CL) was determined from two perpendicular measurements, one of which was the largest diameter. The mean of the two measurements was used as the ovarian (OD), follicular (FD), and CL (CLD) diameter. All examinations were performed by two persons working together 90% of the time. Neither one knew the results of the vaginal smear before the US examination was performed. A decrease of at least 10% of the FD of the dominant follicle (DF) coinciding with the presence of leukocytes in the vaginal smear was considered to be indicative of ovulation. Data Analysis The US recordings were normalized to the first day of the cycle (day 1), the last day of the largest FD (day US-0), or the day of the E2 peak (day E-0). We analyzed the temporal distribution of maximal and minimal myometrial and endometrial thicknesses, according to different intervals relative to E2 peak, by the chi-square test (w2), using the null hypothesis of homogeneous distribution. The mean size of the ovary containing a DF was compared with that of the contralateral one by Student’s paired t-test. The largest FD of three consecutive cycles was compared within and between animals by analysis of variance (ANOVA) for repeated measures. The data are presented as mean7SE, unless otherwise stated. A P-value o0.05 was considered statistically significant. We analyzed the relationships between the size of the uterus and the endometrial thickness, and the DF and concentration of E2, as well as between the largest FD and the size of the early CL using Pearson’s analysis of correlation. We analyzed the probability that the ovulatory follicle would occur in the same ovary in consecutive cycles using the chi-square goodness-of-fit test and the Markov chain model. Both analyses included 22 periods of observation [Mizrahi & Sullivan, 1988]. RESULTS Monitoring the Menstrual Cycle by Vaginal Smears The follicular phase was defined as the period beginning the first day of bleeding and ending the last day of the periovulatory period, both days included. The periovulatory period was defined as the period beginning with a sharp increase of the eosinophilic cells and ending with a decreasing number of these cells associated with the appearance of leukocytes and intermediate cells, which indicated the beginning of the luteal phase. Experiment 1. All of the cycles (n=30) were ovulatory, and exhibited normal duration and sequence of menstruation, follicular, periovulatory, and luteal phase, as indicated by vaginal cytology. The length of the menstrual cycle varied from 16 to 24 days Cebus apella Ovarian Cycle / 237 Fig. 1. US images and outline drawings of the uterus of a cebus monkey. A.1 and A.2 and B.1 and B.2 correspond to images and outlines of the transverse and longitudinal sections, respectively. In A.1 and B.1, the prominent echoic line between x and x is the interface between adjacent layers of the endometrium. In A.2 and B.2, arrows 1 and 3 indicate the dorsoventral thickness of the uterus, whereas a and b indicate the dorsoventral thickness of the endometrium. In A.2, arrow 2 indicates the transverse diameter of the uterus. with a mean value of 19.570.4. The mean length of the follicular and luteal phase was 8.270.2 and 11.370.4 days, respectively. The follicular phase included menstruation lasting 3.570.2 days and a periovulatory period lasting 4.770.2 days. The serum E2 and P levels determined throughout the first and third cycles exhibited levels and oscillations typical of a normal biphasic ovulatory cycle. In all cases a large number of superficial eosinophilic cells in the vaginal smear coincided with the presence of a preovulatory follicle and high levels of E2, while a reduced number of eosinophilic cells associated with leukocytes and intermediate cells coincided with the presence of a CL and/or high levels of P. The uterus and both ovaries were identified in each US examination (n=327). The myometrium and endometrium were clearly distinguished (Fig. 1). The mean maximal and minimal thicknesses of the myometrium were respectively 7.270.2 mm with a range of 6.0–8.7 mm, and 5.270.1 mm with a range of 3.9– 6.6 mm. Variations along the cycle or between cycles were minimal. The endometrium presented a trilaminar conformation (Fig. 1) with little changes along the cycle. The mean maximal and minimal thicknesses across animals were 7.270.2 mm and 5.570.2 mm, with ranges of 5.1–8.8 mm and 3.5–7.0 mm, respectively. 238 / Ortiz et al. TABLE I. Timing of Maximal and Minimal Myometrial and Endometrial Thickness Relative to E2 Peak in 20 Cycles Number of cycles in which extreme thicknesses were observed Maximal thickness Days relative to E2 peak Myometrium 7 to 3 2 to +2 +3 to +7 +8 to +12 +13 to +18 a 6 11 (55%) 2 1 0 a Endometrium 2 13 (65%) 2 0 3 Minimal thickness a Myometrium Endometriuma 3 6 6 2 3 11 (55%) 3 4 2 0 Po 0.05. Fig. 2. US images of the left ovary of a cebus monkey during the menstrual cycle. A–C show the development of the dominant follicle (DF) from middle to late follicular phase, and D shows the CL in the early luteal phase. The uterus (U) is seen adjacent to the ovary in A. When the maximal and minimal myometrial and endometrial thicknesses observed in each cycle were distributed over different 5-day intervals, before and after the E2 peak, a different distribution was found for both structures (Table I). In 11 of 20 and 13 of 20 cycles, respectively, the myometrium and endometrium Cebus apella Ovarian Cycle / 239 exhibited their maximal thicknesses within 2 days before and 2 days after the E2 peak. On the other hand, the minimum myometrial thickness was homogeneously distributed at different intervals before and after the E2 peak. As for the endometrium, in 11 of 20 cycles it exhibited its minimum thickness during a period of five days. This period included from day 7 to day 3 before E2 peak. The DFs were seen as round or slightly ovoid hypoechoic structures with well delimited borders within the outline of the ovaries, during 5.070.3 days (range=2–10 days) (Fig. 2). The progressively increasing size of the DFs was observed in all cycles. The FD of the DFs at the first measurement of each cycle varied from 2.7 to 6.6 mm. During the last 4 days of follicular development, diameters increased at a rate of 0.8–1 mm/day and reached an FD of 8.270.2 mm. The largest FD observed in different cycles of the same animal varied o1.0 mm in seven females, and o1.6 mm in the remaining three. The largest FD recorded across animals yielded a range of 6.4–9.7 mm and differed between animals (P=0.0002), but not between cycles of the same animal. When US recordings of the FD were normalized to the day of the E2 peak (day E-0), a positive correlation of 0.89 (P=0.007) was found between the increasing FD and the concentration of E2 measured until the day of the peak. The largest FD was observed 1 day before (n=4), on the same day (n=3), and 1 day (n=4) or 2 days (n=9) after the E2 peak. In all cycles a decrease by Z10% of the largest FD coincided with the presence of leukocytes in the vaginal smear. In the majority of cycles (25/30), this decrease occurred 1 day after the DF reached the largest diameter. In these cases the decrease was 421% in 17 cycles and 10–20% in eight cycles. In the remaining five cycles, the decrease occurred 2 days after the DF reached the largest diameter, and it was 21% and 25% in two cycles and 10–20% in the remaining three cycles. Two patterns of follicular rupture and voiding were observed. Cases that reached the largest FD on the day of the E2 peak or later, had a decrease in the FD by one-fourth to one-third. This decrease coincided with serum P concentrations of about 600 nmol/L and the appearance of leukocytes in the vaginal smear (Fig. 3a). Cases that reached the largest FD 1 day before the E2 peak had a small decrease (7%) coinciding with P concentrations of about 160 nmol/L. In these cases, leukocytes appeared in the vaginal smear the following day, coinciding with an additional decrease (by 19%) of FD and P concentration that was not lower than 450 nmol/L (Fig. 3b). Throughout the cycle, the ovary containing the dominant follicle was only slightly bigger than the contralateral one. When the DF reached its largest diameter, the size of the ovulatory and nonovulatory ovaries was 11.570.1 mm and 11.0 70.1 mm, respectively. The CLs appear as ovoid images, are less hypoechoic than follicles, and have an irregular border. The US image of the CL remained recognizable, with a rather constant size, during 1–2 days in 12 cycles, 3–4 days in seven cycles, 5–6 days in five cycles, 7–8 days in five cycles, and 13 days in only one cycle. The length of time the CL remained visible was positively correlated (r=0.42; P=0.05) with the FD observed just prior to ovulation. Experiment 2. All of the cycles (n=132) were ovulatory and exhibited normal features. Both ovaries were identified in each US examination (n=150). The side of the DF did not alternate in any of the animals, and both statistical analyses confirmed 240 / Ortiz et al. Follicle P4 Corpus luteum 8 ovulation 1400 7 1200 6 1000 5 800 4 600 3 400 2 200 1 0 0 1 Vaginal (a) cytology 3 Menses 1800 5 7 9 11 13 Days of the cycle Periovulatory period 15 17 18 Luteal phase E2 Follicle P4 Corpus luteum 10 ovulation 9 1600 8 1400 E2 (pmol/L) P4 (nmol/L) 19 7 1200 6 1000 5 800 4 600 3 400 2 200 1 0 0 1 Vaginal (b) cytology 3 Menses 5 7 9 11 13 Days of the cycle Periovulatory period 15 Diameter (mm) E2 (pmol/L) P4 (nmol/L) 1600 9 Diameter (mm) 1800 E2 17 Luteal phase Fig. 3. a and b: Chronologic relationships between the concentrations of E2, P, follicular development, CL, and vaginal cytology during the menstrual cycle of two cebus monkeys, which are representative of the two patterns of follicular rupture and voiding observed in these animals. a: The largest FD was observed 1 day after the E2 peak, and the decrease in the FD coincided with the appearance of leukocytes in the vaginal smear. b: The largest FD was observed 1 day before the E2 peak, and a small decrease in the FD coincided with the E2 peak. In this case, leukocytes appeared in the vaginal smear the following day. Cebus apella Ovarian Cycle / 241 TABLE II. Side of the Leading Follicle in 18 Monkeys Followed For Six Consecutive Cycles Consecutive cycles Female no. 1 8 16 19 47 62 68 109 115 120 125 139 162 233 18 a 18 b 124 a 124 b 140 a 140 b 174 a 174 b 1st 2nd 3rd 4th 5th 6th L R L R R L R L R L R L R R R R R L R L R L R L R R R L L R R R L L L R L L L L R L R R R L L L L R L L L L L L R R L R R R L R L R R R L R L L R L L L L R R R L L L L L R L R R L L L L R L L L R R R R R L R L R L R L L L R L R R L R L L R L L R L L R L R L R R R L, left ovary; R, right ovary; a, b=one year lapse between the two monitoring periods. randomness in the side of ovulation in consecutive cycles. None of the animals developed the DF in the same side for six consecutive cycles, but in 15 of the 22 sets of six cycles (68.2%), ovulation took place in the same side in three to five consecutive cycles. This proportion did not reach statistical significance. Half of the animals monitored in two distant periods exhibited similar patterns of persistence and/or change in the side of the leading follicle in the two periods (Table II). DISCUSSION This study shows that in the New World monkey Cebus apella, the ovaries, follicles, CLs, and uterus can be serially observed and measured under anesthesia throughout the menstrual cycle using transabdominal US, irrespective of urinary bladder volume, and without disturbing the duration and sequence of the phases or the oscillations in the plasma levels of E2 or P as previously described [Nagle & Denari, 1983]. The changes in the serum concentrations of E2 and P, and the relationships between follicular growth/ovulation and vaginal cytology and hormonal parameters were similar to those reported by Nagle et al.  and Nagle and Denari , who used laparoscopy to visualize the ovaries. In their studies and the current one, the presence of a large number of superficial eosinophilic cells in the vaginal smear coincided with the presence of a preovulatory follicle and high levels of E2, while a reduced number of eosinophilic cells associated with leukocytes and intermediate cells coincided with the presence of a CL and/or high 242 / Ortiz et al. levels of progesterone (Fig. 3a and b). We found higher concentrations of P in the mid-luteal phase than previously reported [Nagle et al., 1979; Nagle & Denari, 1983]. The difference may reflect the use of different RIA protocols and/or antibodies in the different studies [Nagle et al., 1979]. In this study we confirmed that E2 and P concentrations in the capuchin monkey, as in other New World monkeys, reach values higher than those reported in women and Old World monkeys. The physiological significance of these differences remains to be elucidated. On the other hand, the plasma levels of LH in serum reported by Nagle and Denari  have not yet been confirmed, and are not reported here because we found that neither commercially available reagents for the immunoassay of gonadotropins nor the Leydig cell bioassay method for LH [Van Damme et al., 1974] are useful for measuring cebus gonadotropins in serum (unpublished results). It is noteworthy that in each of the 162 cycles monitored by US, only a single follicle was observed that finally developed to a preovulatory stage. This finding agrees with the fact that three gestations of monocygotic twins have been observed in 125 pregnancies monitored by US and diagnosed at the time of caesarean section. Interestingly, although they occurred in three different females, two of the females had mated with the same male (unpublished observations). The proportion of 3/125 (2.4%) is identical to that observed in 10 pregnancies that resulted in twin litters out of 425 pregnancies that occurred over a 15-year period (1985–1999). Those pregnancies were recorded in the breeding colonies of cebus monkeys maintained by the Laboratory of Comparative Ethology at the National Institutes of Health Animal Center (NIHAC), Poolesville, MD; the University of Georgia (now maintained by LABS of Virginia, Hampton, SC); Kassel University, Kassel, Germany; and the Instituto di Psicologia, CNR, Rome, Italy [Leighty et al., 2004]. The genetic condition (monozygotic or dizygotic) of twins is not reported in the article by Leighty et al. . Our observations and those reported by Leighty et al.  suggest that in the cebus monkey, normally one follicle reaches the preovulatory stage. This differs from results of observations with US in women where [Gore et al., 1995] reported 15% of cycles exhibiting more than one dominant follicle reaching the preovulatory stage. We assume that the decrease by at least 10% of the FD of the DF coinciding with the presence of leukocytes in the vaginal smear is indicative of ovulation because the relationship between ovulation and the presence of leukocytes in the vaginal smear has been confirmed by direct observation of the ovaries through laparotomy and oocyte recovery from the oviduct [Ortiz et al., 1995]. Nagle et al.  identified the dominant follicle during the periovulatory period using sequential laparoscopies. They described larger preovulatory follicles (10–12 mm) than those observed in the present study (6.4–9.7 mm). With US one can better distinguish between the ovarian stroma and the follicular wall, and determine the size of the follicle from two perpendicular measurements, which may account for the smaller FD observed in our study. This may also account for the smaller difference we observed between the ovulatory and nonovulatory ovaries. The relationship between endometrial thickness and E2 levels and the trilaminar appearance of the uterus is similar to that observed in rhesus monkeys [Morgan et al., 1987] and humans [Gormaz et al., 1992]. However, in contrast to what is observed in macaques and women, whose endometria change from highly anechoic at the late follicular to echoic during the luteal phase, in the cebus monkey the reflectivity of the endometrium is less variable and maintains an almost constant anechoic image throughout the cycle. Cebus apella Ovarian Cycle / 243 The complete lack of regular alternation of the ovulatory side, or randomness of the ovulatory side, negates the occurrence of CL-dependent intra- or interovarian mechanisms designed to alternate the side of emergence of the dominant follicle in this species. On the contrary, there was a tendency to ovulate in the same side in consecutive cycles, as previously reported in women [Werlin et al., 1986], which suggests that a mechanism may operate in both species to facilitate the repetition of ovulation in the same side in consecutive cycles. Undoubtedly, a larger number of subjects and number of consecutive cycles should be followed up to more accurately determine the prevalence of such repetition and the significance of these preliminary observations. 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