Does the conceptus of the viviparous lizard Barisia imbricata imbricata participates in the regulation of progesterone production and the control of luteolysis?

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ANIREP 4972 1–9

Animal Reproduction Science xxx (2014) xxx–xxx 1

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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci

Does the conceptus of the viviparous lizard Barisia imbricata imbricata participates in the regulation of progesterone production and the control of luteolysis?

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Martín Martínez-Torres a,∗ , Martha Salcedo-Álvarez a , Carmen Álvarez-Rodríguez a , Mario Cárdenas-León b , Juana Luis a , Leticia Moreno-Fierros c a Laboratorio de Biología de la Reproducción, Facultad de Estudios Superiores, Iztacala, Universidad Nacional Autónoma de México, Avenida de los Barrios no. 1, Los Reyes Iztacala A. P. 314, Tlalnepantla 54090, Estado de México CP, Mexico b Laboratorio de Hormonas Proteicas, Departamento de Biología de la Reproducción, Instituto de Ciencias Médicas y de la Nutrición Salvador Subirán, México DF, Mexico c Laboratorio de Inmunidad en Mucosas-Unidad de Biomedicina, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Los Reyes Iztacala AP 314, Tlalnepantla CP 54090, Estado de México, Mexico

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Article history: Received 17 June 2013 Received in revised form 5 May 2014 Accepted 9 May 2014 Available online xxx

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Keywords: Viviparity Progesterone Corpus luteum Pseudopregnancy Reptiles Lizards

It is generally accepted that progesterone is necessary to maintain gestation; however, the mechanisms that control the production of this steroid remain unknown. The corpus luteum has been assigned a central role in the maintenance of gestation based on its capacity to produce progesterone. A pseudopregnancy model was performed in a viviparous lizard, Barisia imbricata imbricata, to determine whether the absence of embryos would affect the pattern of progesterone production or the corpus luteum histology. Blood samples were obtained prior to ovulation and at 8, 16, and 24 weeks after ovulation (pseudopregnant and pregnant lizards), as well as one day after parturition (pregnant lizards) or 32 weeks after ovulation (pseudopregnant lizards). The corpus luteum was surgically removed one day after blood samples were obtained. Blood aliquots from nongravid females were obtained at similar timepoints. We found a significant reduction in plasma progesterone concentrations at 24 and 32 weeks post-ovulation in pseudopregnant lizards compared with those observed at similar times in intact pregnant lizards, whereas the progesterone levels in non-gestant lizards remained significantly lower than in either pseudopregnant or pregnant lizards. Moreover, we observed that the histological appearance of the corpus luteum from pseudogestational females (obtained 24 and 32 weeks post-ovulation) differed from the corpora lutea from lizards in late gestation and intact parturient lizards. These observations suggest that the conceptus participates in the regulation of progesterone production in late gestation and also in luteolysis control. © 2014 Elsevier B.V. All rights reserved.

∗ Corresponding author. Tel.: +0052 55 5623 1258; fax: +0052 55 5623 1155. E-mail address: [email protected] (M. Martínez-Torres). http://dx.doi.org/10.1016/j.anireprosci.2014.05.003 0378-4320/© 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: Martínez-Torres, M., et al., Does the conceptus of the viviparous lizard Barisia imbricata imbricata participates in the regulation of progesterone production and the control of luteolysis? Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.05.003

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1. Introduction

Viviparity is a reproductive strategy utilised by many 32 species in most major clades of vertebrates, (Angelini and 33 Ghiara, 1984; Rothchild, 2003). Several phylogenetic analy34 ses indicate that this trait evolved independently more than 35 100 times within Squamata, a frequency greater than that 36 of all other vertebrate clades (Blackburn, 1995; Wang and 37 Evans, 2011). Paleontological and embryological evidence 38 indicates that oviparity in reptiles represents a primitive 39 reproductive strategy that led to viviparity (Blackburn, 40 1982; Ghiara et al., 1987). During the transition to vivipar41 ity, anatomical and physiological modifications affected 42 the gestant females and conceptuses. These modifications 43 included the following: (1) extension of the lifespan of the 44 corpus luteum (CL), (2) enlargement of the secretion pat45 tern of progesterone (P4 ), (3) increased uterine vascularity, 46 (4) reduction in the number, size, and activity levels of 47 the uterine glands, (5) loss of calcareous shell, and (6) pla48 cental development (Blackburn, 1998; Callard et al., 1992; 49Q2 Guillette, 1993, 1995 Guillette and Jones, 1985; Wourms 50 and Callard, 1992). 51 The need for P4 to sustain gestation is well established; 52 no species has yet been discovered that does not require 53 the steroid for the maintenance of pregnancy (Guillette 54 et al., 1985, 1989; Bourne et al., 1986; Xavier, 1987; Callard 55 et al., 1992; Martínez-Torres et al., 2003). In mammals, 56 several lines of evidence show that the P4 is crucial in 57 the maintenance of gestation (Graham and Clarke, 1997). 58 The major physiological role of this hormone during preg59 nancy in this vertebrates, is the maintenance of pregnancy 60 by the promotion of uterine growth and the suppression 61 of myometrial contractility (Yaron, 1972, 1985; Graham 62 and Clarke, 1997; Mulac-Jericevic and Conneely, 2004). 63 In reptiles, P4 has been detected in gravid females in all 64 stages of pregnancy (for review, see Xavier, 1987) and, 65 although the profiles of this hormone vary dramatically 66 among viviparous squamate species (Jones and Baxter, 67 1991; Xavier, 1987), several studies agree that the P4 may 68 play a central role in embryo retention during gravidity and 69 in the evolution of viviparity (Callard et al., 1992; Guillette, 70 1985; Shine and Guillette, 1988; Yaron, 1985; Martínez71 Torres et al., 2010). 72 On the other hand, there are studies that show, 73 that in the majority of viviparous lizards, the CL is a 74 principal source of P4 during gestation (for review see 75 Xavier, 1987). This finding has led several authors to 76 assign a central role in the maintenance of gestation and 77 the evolution of viviparity to this gland. However, sev78 eral lines of indirect experimental evidence suggest that 79 viviparous squamata possess a secondary source of P4 . 80 In Lacerta vivipara, Dauphin-Villemant and Xavier (1985) 81 and Dauphin-Villemant et al. (1990) observed an in vitro 82 increase in the adrenal activity and production of P4 dur83 ing gestation. In Chalcides chalcides, Sceloporus jarrovi and 84 Barisia imbricata, it has been observed that the placenta is 85 capable of producing (Guarino et al., 1998; Martínez-Torres 86 et al., 2006a) and metabolising P4 (Painter and Moore, 87 2005). Moreover, lutectomy or ovariectomy in early preg88 nancy does not completely eliminate the plasma P4 in the 89 viviparous lizards Tiliqua rugosa (Fergusson and Bradshaw, 31

1991) and B. i. imbricata (Martínez-Torres et al., 2010) or in viviparous snakes (Thamnophis sirtalis, Highfill and Mead, 1975). Participation of the conceptus in the prevention of luteal tissue regression and in the regulation of P4 production by CL has been widely documented in several mammalian species (Bazer and Roberts, 1983; Gordon and Net, 1988; Gordon et al., 2000; Zeleznick and Clifford, 2006). A simi- Q3 lar phenomenon may occur in reptiles. Xavier et al. (1989) found indirect evidence that suggest that the conceptus of L. vivipara could participate in the regulation of P4 production and CL activity. B. i. imbricata is a Mexican viviparous temperate lizard, commonly known as the scorpion or alligator Popocatepetl lizard, which exhibits autumnal reproduction (Guillette and Casas-Andreu, 1987; Martínez-Torres et al., 2003). Oogenesis occurs during summer and fall, and ovulation occurs during November or early December (MartínezTorres et al., 2006a). Mating takes place several weeks before ovulation occurs (Martínez-Torres, unpublished data). In this species, gestation occurs throughout the winter months and most of the spring (from late November or early December until late May or early June) (MartínezTorres et al., 2003). The CL develops during the first third of pregnancy, and luteolysis occurs during the remaining months. Four sequential stages have been identified during luteal development and three stages during luteal regression (Martínez-Torres et al., 2003). It has been observed that there is a positive correlation between the P4 plasma levels and the histological appearance and histochemical activity of 5−4 -isomerase-3␤ hydroxy steroid dehydrogenase in the luteal tissue (Martínez-Torres et al., 2003) throughout gestation. These observations suggest that the CL is the major source of P4 during pregnancy in B. i. imbricata. However, in this lizard, it was recently observed that ablation of the CL in early pregnancy provoked a significant reduction in P4 plasma levels during the first half of pregnancy without inducing abortion but resulting in abnormal parturition (Martínez-Torres et al., 2010). Despite the importance that several researchers have assigned to P4 in the maintenance of gestation and the evolution of reptilian viviparity, the mechanisms that modulate the production of this hormone (from corpus luteum or an extra-ovarian source) remain unknown. In this paper, a pseudopregnancy model was used to determine whether the absence of a conceptus modifies the plasma P4 levels and the appearance of the CL in the viviparous temperate lizard B. i. imbricata.

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2. Materials and methods

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2.1. Animals

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Adult females (113 ± 36 mm snout-vent length and 24.6 ± 5.7 g body weight) of B. i. imbricata were collected after the mating period but before ovulation (the last week of October and first week of November in 2007, 2008 and 2010) from Cuautitlán, México State (19◦ 37 N, 99◦ 11 W; 2253 m altitude). Females were toe-clipped for individual identification. On the day of capture, all lizards collected were transported to the laboratory and submitted for


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sperm detection by means of vaginal flushing (MartínezTorres, 2009). Ultrasound was performed 2–3 days after capture, using an ACUSON ASPEN unit with a 7–10 MHz 150 transducer to determine the size of the vitellogenic follicles. 151 Lizards that had vitellogenic follicles of 8.5–9.0 mm (pre152 Q4 ovulatory follicles, POF, Martínez-Torres et al., 2006a,b) 153 were randomly assigned to the following study groups: (i) 154 pseudopregnant lizards (PPL: n = 23); (ii) pregnant intact 155 lizards (PIL: n = 25) (iii) negative control (NC, non-pregnant 156 157 lizards: n = 16). 148 149

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2.2. Pseudopregnant and negative control lizards Tubal ligation was performed on lizards one day after the detection of POF. The procedure was conducted as follows: the females were anesthetised with ether, and a ventrolateral incision was made; the uterus was immediately located, and a ligature was placed at the boundary between the infundibulum and uterus. The ligation was open enough to allow the sperm to access the infundibulum but closed enough to prevent passage of eggs to the uterus. On the day of surgery and the following day, each lizard received an injection (I.M.) of 5000 U.I. of penicillin G and 500 ␮g streptomycin sulphate. Lizards were maintained in captivity in individual terraria with free access to water and food (mealworms, grasshoppers and crickets in the greenhouse (2.60 m × 5.00 m × 2.70 m) at the UMF-FES Iztacala UNAM (19 ◦ C 31 N; 98 ◦ C, 11 W; 2250 m altitude). Temperature was not controlled and oscillated from 0 ◦ C (minimal temperature registered during the winter) to 34 ◦ C (maximal temperature registered during the spring); the photoperiod was natural. Lizards were scanned weekly by ultrasound to determine whether ovulation had taken place (Martínez-Torres et al., 2006a). Once ovulation was detected, the females were submitted to laparotomy to remove the eggs or vitellus from the abdominal cavity; ligatures were also removed. These females were considered to be pseudopregnant lizards (PPL). In lizards that did not ovulate, the ligatures were removed, and the lizards were assigned to the negative control group (NC). After egg removal and/or ligature removal, the lizards were sutured and deposited in a terrarium with free access to water and food for the following three days. Each lizard (PPL and NC) received an injection (I.M.) of 5000 U.I. of penicillin G and 500 ␮g streptomycin sulphate. After the recovery period, all PPL and NC lizards were liberated to a greenhouse and maintained with free access to water and food throughout the experiment under the environmental conditions described above. 2.3. Pregnant intact lizards

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The PIL did not undergo surgical treatment; however, weekly scanning was performed to identify the timing of ovulation. These females received antibiotics and were maintained in the same conditions as the PPL throughout the experiment.

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2.4. Plasma collection

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Blood samples of 200–220 ␮l were obtained from each lizard (PPL, NC, PIL) by cardiac puncture with a heparinised

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syringe. In this study, the first sample was obtained immediately after the lizards were collected (before ovulation). Blood samples were then centrifuged at 10,000 rpm for 30 s; the plasma was decanted into an eppendorf tube and stored on dry ice. On the same day, samples were transported to the laboratory and stored at −70 ◦ C until the radioimmunoassay (RIA) for P4 was performed. Only the aliquots from lizards with POF were included in this study (n = 16) for determination of the P4 plasma concentrations prior to ovulation; the remaining samples were discarded (i.e., from lizards with follicles less than 8.5 mm). The aliquots from PIL were obtained at 8 (early gestation, n = 5), 16 (middle gestation, n = 5) and 24 weeks after ovulation (late gestation, n = 5), as well as one day after parturition (immediately post-parturition, n = 5). Blood samples were obtained at the same time points for PPL [8 (n = 5), 16 (n = 4), 24 (n = 5) and 32 weeks after ovulation (n = 4)]. Each lizard was sampled 2 times, the first sample at time of capture (prior to ovulation), the second at one time point after ovulation. Finally, we obtained blood samples of similar volumes and at similar timepoints from the NC group (n = 16). 2.5. Corpus luteum Partial lutectomy was performed one day after blood samples were obtained from PIL in the early, middle and late stages of gestation and during the ipP time period. Treatment was performed in the PPL group using the same time points as in the PIL group. The procedure was as follows: the lizards were anesthetised with ether, a ventrolateral incision was made, and the right ovary was located. One or two CL were surgically removed from each lizard and fixed in 10% buffered formalin for 24 h. The CL were dehydrated in a graded alcohol series, processed according to routine histological technique and embedded in Paraplast (Luna, 1968). Histological sections (7 ␮m thick) from luteal tissue were stained with hematoxylin and eosin and were observed under the microscope to characterise their histological appearance and identify the stage of development or regression of luteal tissue according to Martínez-Torres et al. (2003). All experimental procedures were approved by the Bioethical Committee of the FES Iztacala UNAM. 2.6. Radioimmunoassay Progesterone concentration in the plasma was analysed in duplicate without prior dilution or extraction using a commercial radioimmunoassay kit (Coat. A-Count Progesterone, Diagnostic Products Corporation, CA, 90045). 125 I-labelled progesterone was supplied as the reactive tracer, and the antiserum was specific for P4 . Steroids showing cross-reactivity (relative to progesterone, 100%) were androstenediol (non-detectable), corticosterone (0.9%), cortisol (0.03%), 11-deoxicorticosterone (2.2%), 20adihydroprogesterone (0.2%), estradiol (non-detectable), 17␣-hydroxyprogesterone (3.4%), 5b-pregnan-3a-ol-20one (0.05%), 5a-pregnan-3, 20-dione (3.2%), pregnenolone (0.1%), and testosterone (0.1%). The inter- and intra-assay coefficients of variation were 9.2% and 8.6%, respectively.


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The sensitivity of the assay was 0.12 ng/ml, and the accuracy of the assay was determined according to Bourne et al. (1986), by adding various concentrations (within the normal assay range) of P4 to the plasma. Linear regression analysis of the data yielded a correlation coefficient of 0.999. We validated the assay through parallel comparison of a serial dilution of plasma pooled from pregnant female B. i. imbricata (r2 = 0.95). The values shown in the results were obtained using GAMBYT software.

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The mean gestation time in PIL B. i. imbricata was estimated to be 31.7 ± 2.6 weeks. Ovulation occurred during November (between November 1 and November 26), and parturition occurred during the period from June 1 until July 18.

tissue of PIL demonstrated advances in the luteolytic process (two lizards in stage II of regression, three lizards in stage III of regression), the CL from all pseudopregnant lizards showed a histological appearance that resembled stage II of luteolysis. That is, the CL in stage II from PIL displayed cells with shrunken or broken nuclei, ring cells, abundant infiltration of connective tissue, and thinning thecal tissue (Fig. 1E). The luteal cell mass from CL in stage III of PIL included some cells whose nuclei were shrunken or broken, abundant hypertrophied, ring cells, infiltration of trabeculae of connective tissue with small blood vessels and some small and round or irregular cavities (Fig. 1F). In contrast, the luteal cell mass in PPL was composed mainly of ring cells and a few trabeculae of connective tissue with wide blood vessels (Fig. 1G). The luteal tissue from ipP exhibited changes characteristic of the final stage of luteal regression. The LCM exhibited a reticular aspect and some connective tissue trabeculae. We observed that the CL from 2 PIL also showed small, irregular cavities. Different cell types were observed in the luteal tissue of all PIL. All cells showed basophilic nuclei with condensed chromatin and scant cytoplasm, but the nuclei showed diversity in size and form, ranging from ovoid to irregular shapes. We also observed the infiltration of eosinophil leukocytes (Fig. 1H). In contrast, we found that the CL of all PPL from 32 weeks post-ovulation continued to display ring cells (hypertrophied and small), and some eosinophils were also observed in the luteal tissue. In addition, we observed several rounded or elongated cavities and large blood vessels (Fig. 1I).

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3.1. Corpus luteum

3.2. Progesterone

The histological appearance of luteal tissue obtained at 8 and 16 weeks from PPL was similar to that of the CL during early and mid-gestation. However, histological differences were observed in CL obtained at 24 and 32 weeks postovulation compared with CL from intact pregnant lizards at equivalent timepoints (late gestation and ipP, respectively). All CL obtained at 8 weeks post-ovulation (from PPL and PIL) displayed the histologic appearance of mature CL (stage IV according to Martínez-Torres et al., 2003). The luteal cell mass (LCM) displayed only oval cells with acidophilic cytoplasm and basophilic nuclei (Fig. 1A). We also observed some thin trabeculae of connective tissue with small capillaries within the LCM. The luteal tissue of PPL displayed a similar appearance (Fig. 1B). In contrast, all CL from 16 weeks post-ovulation, from both groups, showed typical characteristics of luteal regression (Fig. 1C and D, respectively). The luteal cell mass from PIL and PPL displayed “ring cells” (oval cells with lipid vacuoles, scant acidophilic cytoplasm, and flattened basophilic peripheral nuclei). Moreover, several thin trabeculae of connective tissue were also observed in the LCM (Fig. 1C and D). We observed that the only differences between PIL and PPL were the thickness and, to a lesser extent, the abundance of trabeculae; both were higher in the PPL group. This histological appearance corresponds to stage II of luteolysis. The luteal tissue obtained from PPL 24 weeks after ovulation showed histological differences from the CL obtained during the late gestation period. Whereas the luteal

We observed similar patterns of P4 levels between PPL and PIL. There was an increase at mid-gestation, followed by a gradual decrease during the remaining gestation time. In contrast, the NC females showed significantly lower concentrations throughout the study (Fig. 2). Therefore, the rest of our comparisons of progesterone levels were evaluated solely between PPL and PIL. We found that the absence of embryos did not significantly alter the concentrations of plasma P4 in times equivalent to early [F(2,4) = 1.4, p = 0.16; PPL = 1.85 ± 0.10 ng/ml vs. PIL = 2.12.4 ± 0.26 ng/ml] or mid-pregnancy [F(2,4) = 2.5, p = 0.09; PPL = 1.93 ± 0.23 ng/ml vs. PIL = 2.42 ng/ml ± 0.33 ng/ml] (Fig. 2). However, there was a significant decrease in plasma P4 in lizards lacking embryos during the period equivalent to late gestation [F(2,4) = 3.2, p < 0.05; PPL: 0.43 ± 0.18 ng/ml vs. PIL: 0.82 ± 0.16 ng/ml] and the immediate postpartum period, in comparison to intact pregnant lizards (F(2,4) = 3.2, p < 005; PPL: 0.26 ± 0.14 ng/ml vs. PIL: 0.50 ± 0.12 ng/ml; p < 0.05) (Fig. 2).

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The P4 data were analysed using a two-way analysis of variance for repeated measures (ANOVA) (Zar, 1974). Posthoc tests were carried out using the Holm–Seldak method to determine whether differences in plasma progesterone levels between PPL, PIL and NC existed at various postovulation time points. Results were considered significant at p < 0.05, and all values shown are means ± SEM. All statistical analyses were performed with Sigma Stat software (3.5 for Windows).

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3. Results

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4. Discussion Viviparity requires that the egg be retained in the uterus until embryonic development is completely finished; consequently, it is essential to maintain a hormonal milieu that will allow gestation to proceed appropriately (Amoroso, 1969; Guillette, 1987). Although reptilian Q5


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Fig. 1. Histological sections of corpus luteum (CL) from pregnant intact. lizards (PIL) and pseudopregnant lizards (PPL) of Barisia imbricata imbricata. (A) and (B) Histological sections from PIL and PPL, respectively, at 8 weeks post-ovulation. The CL in (A) and (B) are in stage IV of luteal development. Note the presence of cells with light acidophilic cytoplasm and nuclei possessing one, two or three nucleoli ( ) and cells with nuclei with condensed chromatin ( ). Scale bar, 25 ␮m. In (C) and (D), histological sections from PIL and PPL, respectively, at 16 weeks post-ovulation. Note the presence of “ring cells” ( ) and trabeculae of connective tissue (t). Scale bar, 50 ␮m. (E) Histological section from PIL at 24 weeks post-ovulation (stage II of regression). The regression continues; note the presence of scanty “ring cells” ( ) and the cells with shrunken (→) or broken ( ) nucleus in the luteal cell mass. Scale bar, 50 ␮m. (F) Histological section from PIL at 24 weeks post-ovulation (stage III of regression). The luteolysis advances: note the presence of hypertrophied ring cells ( ) in cavities in the luteal cell mass as well as thin trabeculae of connective tissue. (G) Histological section from PPL at 24 weeks post-ovulation. Note that only “ring cells” ( ) are present in the luteal cell mass and blood vessels (bv). The histologic appearance resembles stage II of regression. Scale bar, 50 ␮m. (H) Histological section from CL of a lizard, obtained immediately postpartum. The regression continues. Note the presence of blood vessels (bv) and several cellular types as eosinophils (e), ring cells (rc), cells with shrunken nucleus (sn) or cells with condensed chromatin (ccn). Scale bar, 25 ␮m. (I) Histological section from PPL at 32 weeks post-ovulation. Note the presence of “ring cells” [hypertrophied ( ) and small (→)], blood vessels (bv) and small cavities (*). Scale bar, 50 ␮m. Abbreviations nu: nucleoli; sn: shrunken nucleus; rc: ring cell; c: cavity; t: trabeculae; bv: blood vessels; gcn: cells with great condensed nucleus; ccn: cells with clear nucleus; scn: small condensed nucleus; el: eosinophil leukocyte.

viviparity has drawn the attention of numerous authors, most of them have focused their studies on the various ecological pressures that promoted the evolution of 375 this reproductive strategy (Weekes, 1933; Sergeev, 1940; 376 Packard et al., 1977; Tinkle and Gibbons, 1977; Shine 377 Q6 and Bull, 1979; Guillette et al., 1980; Blackburn, 1982; 378 Shine, 1985, 1995, 2002; Ghiara et al., 1987; Andrews, 379 2000; Jian-Fang et al., 2010), and only a few researchers 380 have addressed the proximal mechanisms responsible for 381 the egg retention (Guillette, 1985; Guillette and Jones, 382 1993; Xavier et al., 1989; Callard et al., 1992; Martínez383 Torres et al., 2010). Consequently, information about 384 the mechanism regulating the endocrine medium that 385 373 374

maintains the gestation in viviparous reptiles is scarce. Data obtained in the present study show that, during the first half of gestation, P4 production and luteal development are independent of the embryo. However, we found evidence suggesting that, in late gestation, the conceptus participates in the regulation of P4 production and luteolysis control in the viviparous lizard B. i. imbricata. In mammals, the mechanisms that regulate the hormonal environment during gestation have been well studied (Bazer and Roberts, 1983; Gordon and Net, 1988; Zeleznik and Benyo, 1994; Zeleznick and Clifford, 2006). It is well documented that P4 during pregnancy comes from one or two sources, the luteal tissue and the


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Pattern of progesterona plasma concentration in pseudopregnt, intact pregnant and no ovulated females of the viviparous lizard Barisia imbricata imbricata

CONCENTRATION (ng/ml)

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Fig. 2. Pattern of progesterone plasma concentrations in pregnant (PIL) pseudopregnant (PPL) and non-pregnant lizards (NC) of the viviparous saurus Barisia imbricata imbricata. bO: before ovulation; 8 waO: 8 weeks after ovulation; 16 waO: 16 weeks after ovulation; 24 waO: 24 weeks after ovulation; 32 waO: 32 weeks after ovulation; iPP: immediately pospartum. c is significantly lower with respect to a, but it is higher statistically that e at p < 0.05 d is significantly lower with respect to b at p < 0.05.

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placenta, depending on the species in question (Gordon et al., 2000; Petraglia et al., 2006; Miller and Auchus, 2011). In viviparous reptiles, several findings suggest that the CL is the principal source of this steroid during gestation (for reviews, see Yaron, 1972, 1985; Xavier, 1987; Callard et al., 1992). However, some experimental studies have shown that this gland is not the sole origin of P4 during pregnancy. The adrenal gland and placenta are potential sources of this hormone during gestation in several viviparous squamata (T. sirtalis, Highfill and Mead, 1975; T. rugosa, Fergusson and Bradshaw, 1991; C. chalcides, Guarino et al., 1998; B. i. imbricata, Martínez-Torres et al., 2010). Numerous studies have shown that the conceptus actively participates in the regulation of P4 production from luteal tissue and in the increased lifespan of the CL in several species of mammals, including rats, humans, and mice (Spencer and Bazer, 2004). In contrast, in reptiles, only one paper has been published on this issue. Xavier et al. (1989) found that, in the lizard L. vivipara, the embryo does not stimulate luteal P4 synthesis. In our study, we found that PPL lacking embryos do not differ in serum P4 concentration during the first 16 weeks after ovulation compared to pregnant lizards at equivalent gestational times. This observation shows that, in contrast to mammals, in the viviparous lizards (as B. i. imbricata and L. vivipara), the conceptus does not participate in the regulation of P4 production, either ovarian or extraovarian in origin, during the first half of pregnancy. Moreover, in several mammalian species, in the context of unsuccessful mating or early abortion, the females enter a pseudopregnant stage (Bennett and Vickery, 1970). CL formation occurs in pseudopregnant females, but luteal regression takes place earlier than in normal pregnant

females (as in rabbits) or lasts as long as a normal pregnancy (as in ferrets) (Bennett and Vickery, 1970; Davis and Ryan, 1972; Gordon and Net, 1988; McCraken et al., 1999). In L. vivipara, embryo removal in early pregnancy Q7 causes early luteolysis. However, in our study, we found that in PPL, the luteal development and early luteal regressive changes occurred along a time-course similar to that observed in PIL. These observations show that, in the alligator Popocatepetl lizard, as in pseudopregnant mammals, the presence of an embryo is not necessary for the CL formation, unlike in L. vivipara, where the embryo is involved in the control of luteal development (Xavier et al., 1989). Moreover, unlike mammals, the conceptuses of B. i. imbricata do not prolong the life span-life of the CL because luteolysis begins at the same time in the intact pregnant lizards. The regressive changes during luteolysis, as well as the diminution in P4 plasma concentrations, have been widely documented in reptiles (for review, see Xavier, 1987; Guillette, 1987, 1989). However, few studies have attempted to explain the mechanisms controlling the luteolysis and regulation of P4 production during luteal regression. We found two important changes in PPL during corresponding times in the last third of gestation: (a) the alteration in the luteolytic process and (b) a significant diminution of P4 plasma levels compared to PIL. In our study, we found that in PPL, the luteal development and early luteal regressive changes (i.e., “ring cells” formation) occurred along a time-course similar to that observed in PIL. However, at times corresponding to late gestation, the luteolytic process was disturbed. At 24 and 32 weeks post-ovulation, it was observed that the mass of luteal cells derived from the CL of PPL consisted strictly of “ring cells”, scant connective tissue and some irregular cavities. This cellular type is typical of stage II luteolysis. By contrast, in the PIL group, luteal regression continued to advance gradually (ring cells, cells with shrunken or broken nucleus, cavities), consistent with Martínez-Torres et al. (2003). In mammals, the start of luteolysis is caused by prostaglandin F2alpha (PGF2␣) release from the endometrium of gestant females (McCraken et al., 1999). Guillette et al. (1984) showed that, in the gravid oviparous lizard Anolis carolinensis, the administration of 1.0 ␮g of PGF2␣ caused the diminution of P4 plasma levels and luteolysis. Furthermore, it has been found that the reproductive tract of the viviparous lizard S. jarrovi produces prostaglandins (PGs) such as PGF2␣ and PGE after stimulation by argininevasotocin (AVT) (Guillette et al., 1990, 1992). In this spiny lizard, PG production by the uterus increases in late pregnancy; however, the luteolytic activity of this hormone has not been demonstrated in viviparous lizards. If we accept that uterine PGF2 ␣ also induces luteolysis in viviparous lizards, we could assume that the absence of embryos changes the pattern of PG production in the uterus of pseudopregnant alligator lizards. This phenomenon could be the cause of the alteration in the luteolytic process in PPL. Although the histologic appearance of luteal tissue in the last third of gestation of CL in PPL could be considered an extension of the regressive process, we suspect that these changes represent instead a modification to the luteolytic process because the histological characteristics


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exhibit some important differences from the CL of PIL. It is necessary to continue the study to determine what eventually happens to the corpus luteum of PPL and to measure the PGF2␣ levels to understand their contribution to this situation. The significant reduction of the P4 concentration in late pregnancy was the second change observed in PPL in our study. We considered three possible mechanisms by which P4 might be reduced: (a) alteration of the luteolytic process, (b) removal of the conceptus, and (c) alteration of the activity of the adrenal gland. (a) Alteration of the luteolytic process. It is possible that the disruption of the luteolytic process in PPL may provoke a decrease in the P4 plasma concentration through an increase in the biotransformation of this hormone into other steroids (such as testosterone or E2 ) inside the luteal tissue, as a consequence of an alteration in PG production. This possibility is supported by the following arguments: In the majority of viviparous lizards (Xavier, 1987), it has been observed that the luteolytic process coincides with a gradual diminution of P4 . In A. carolinensis, an oviparous lizard, the administration of PGF2␣ reduced the P4 production by CL as an initial physiological change of luteolysis (Guillette et al., 1984). Moreover, in viviparous saurians, it has been observed that, while the P4 concentration decreases in plasma in late pregnancy, the 17-␤ estradiol level increases (E2 ) (Niveoscincus mucronatus, Girling et al., 2002; Tiliqua nigrolutea, Edwards and Jones, 2001). In B. i. imbricata, the P4 levels decrease gradually during the second half of pregnancy, and this diminution coincides with the advance of luteolysis; however, the E2 pattern is unknown. In mammals, increasing E2 production at the time of parturition promotes contractility of the uterus, induces uterine receptors for uterotonic agonists (e.g., oxytocin), suppresses receptors for uterotonic inhibitors (e.g., ␤-adrenergic compounds), and increases prostaglandin production (Gibb et al., 2006). Some authors suggest a similar role for E2 in reptilian species because the arginine vasotocin (AVT, uterotonic agent in lizards) is regulated by steroids of ovarian origin, such as P4 and E2 (Guillette and Jones, 1980; Guillette, 1985; Guillette et al., 1991; Fergusson and Bradshaw, 1991). Although it remains unknown whether the P4 lutea may be biotransformed to oestrogen inside the luteal tissue, we hypothesised that, in late pregnancy of the alligator Popocatepetl lizard, luteal P4 may be the precursor to oestrogens for the start of parturition, similar to the case observed in mammals. If this relationship is correct, the alteration in luteolysis may have caused an increase in the P4 metabolism of other steroids (such as testosterone or E2 ) and, consequently, may have caused the significant decrease in P4 plasma concentrations in PPL. However, clarification of this model requires measuring the capacity for P4 and E2 production in the luteal tissue of IPL, as well as in PPL. Alternatively, in a previous paper, we found that that lutectomy did not significantly alter the P4 plasma concentrations in late pregnancy in B. i. imbricata but caused abnormal parturition (Martínez-Torres

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et al., 2010). These observations suggest that the luteal P4 may be an important source of oestrogens in late pregnancy that are necessary for the start of the parturition process. (b) Conceptus absence. During the embryonic development of viviparous reptiles, the embryo develops several placental types (Yaron, 1985; Stewart and Blackburn, 1988), and in some lizards it has been shown, directly or indirectly, that the placenta has the capacity to produce or metabolise steroid hormones (Guarino et al., 1998; Painter and Moore, 2005; Martínez-Torres et al., 2006b). In C. chalcides, the chorioallantoic placenta produces P4 during late pregnancy, when the CL is in the advanced stages of luteolysis (Guarino et al., 1998). Painter and Moore (2005) have shown in vitro that the placental tissue of S. jarrovi is able to clear P4 and cortisol and to biotransform them into other metabolites. Previous work from our group (Martínez-Torres et al., 2006b) found that the omphaloplacental residue of newborn B. i. imbricata stained positive for 5−4 3␤-HSD activity. In this species, the omphaloplacenta and allantoplacenta form when luteolysis is initiated (Martínez-Torres, 1997); however, it remains to be determined when the omphaloplacenta begins endocrine activity and whether the allantoplacenta is also able to synthesise steroids. These observations suggest that the omphaloplacenta of the alligator Popocatepetl lizard is capable of producing or metabolising P4 . Thus, the decrease in the plasma P4 concentration in the PPL may be directly attributable to the absence of the conceptus. (c) Alteration of adrenal gland activity. Several indirect and direct results in viviparous lizards (L. vivipara, DauphinVillemant and Xavier, 1985; Dauphin-Villemant et al., 1990; T. rugosa; Bourne et al., 1986; N. mucronatus, 2003), including B. i. imbricata, (Martínez-Torres et al., 2010), suggest that the adrenal gland may actively participate in P4 production during gestation. In these species (Dauphin-Villemant and Xavier, 1985; Dauphin-Villemant et al., 1990; Bourne and Seamark, 1972; Fergusson and Bradshaw, 1991), lutectomy or Q8 ovariectomy causes the concentrations of this hormone to decrease significantly after surgical treatment, but they do not decrease to undetectable levels. Moreover, in our study, we found that the NC lizards show P4 in plasma beyond 32 weeks, although at low concentration. These observations suggest that, in B. i. imbricata, the adrenal gland may also produce this steroid and that, during gestation, there is a mechanism that regulates the adrenal gland activity, in which the embryo may actively participate. Thus, in PPL, the absence of the conceptus may modify the regulation of the adrenal gland activity and reduce the P4 production from this gland. Currently, however, the mechanism that would regulate the activity of the adrenal glands during pregnancy is unknown. 5. Conclusion Several lines of evidence show that the CL and other extraovarian structures (as adrenal gland and/or placenta)


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in lizards provide P4 for the maintenance of gestation. However, the mechanisms that control the production of this steroid remain unknown. In this paper, we found evidence suggesting that the conceptus participates in the regulation of P4 production and in the control of luteolysis. Based on our observations, we conclude the following: (1) In the viviparous lizard B. i. imbricata, the CL formation and normal development of luteal tissue are independent of the presence of the conceptus. (2) Unlike many mammalian species, the conceptus of this lizard does not increase the half-life of the CL. (3) The conceptus is involved in the control of luteolysis. (4) The conceptus participates in the regulation of progesterone production in late pregnancy. These observations suggest that the modulation of luteolysis may be a key event in reptilian viviparity.

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Conflicts of interest statement

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None. The corresponding author asserts that the co-authors have read the final manuscript, agree with its contents and consent to joining the list of authors of this paper.

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Callard and Hirsch (1976), Shanbhag et al. (2001). Acknowledgements

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This study was partiality supported by grant PAPCA 2006–2007, PROJECT 10 to MMT and IN-212113 PAPITUNAM to JL. We thank Gloria Sánchez, Carolina Espinosa de los Monteros, Beatriz Rubio and staff to Laboratorio de Microscopía-FES Iztacala UNAM. This research partiality fulfilled the requirements for MMT’s Ph.D. in Biology.

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Plasma arginine vasotocin, progesterone, and luteal development during pregnancy in the viviparous lizard Tiliqua rugosa.

Ghrelin and leptin modulate the feeding behaviour of the hawksbill turtle Eretmochelys imbricata during nesting season.

Exogenous progesterone or indomethacin delays parturition in the viviparous lizard Sceloporus jarrovi.

Altitude and prevalence of Tinea imbricata in New Guinea.

A case of tinea imbricata in an Italian woman.

Immunological changes during pregnancy in the viviparous lizard, Chalcides ocellatus.

Phenolic Profiling and Evaluation of Contraceptive Effect of the Ethanolic Extract of Salsola imbricata Forssk. in Male Albino Rats.

Levels of testosterone in the plasma and testis of the viviparous lizard (Lacerta vivipara Jacquin) during the annual cycle.

First record of hybridization between green Chelonia mydas and hawksbill Eretmochelys imbricata sea turtles in the Southeast Pacific.

Genetic structure and natal origins of immature hawksbill turtles (Eretmochelys imbricata) in Brazilian waters.

An experimental study of the gestation costs in a viviparous lizard: a hormonal manipulation.

Oogenesis and ovarian histology in two populations of the viviparous lizard Sceloporus grammicus (Squamata: Phrynosomatidae) from the central Mexican Plateau.

Hepatoprotective effect of Rhodiola imbricata rhizome against paracetamol-induced liver toxicity in rats.

In vitro thermal effects on embryonic cells of endangered hawksbill turtle Eretmochelys imbricata.

Unravelling the microbiome of eggs of the endangered sea turtle Eretmochelys imbricata identifies bacteria with activity against the emerging pathogen Fusarium falciforme.

Functional luteolysis in the rhesus monkey: ovarian estrogen and progesterone during the luteal phase of the menstrual cycle.

Plasma biochemical and PCV ranges for healthy, wild, immature hawksbill (Eretmochelys imbricata) sea turtles.

Development of the conceptus in the bovine.

In vitro uterine contractions in the viviparous lizard Tiliqua rugosa: effects of gestation and steroid pretreatment in vivo.

The Human Antimicrobial Protein Calgranulin C Participates in Control of Helicobacter pylori Growth and Regulation of Virulence.

The regulation of ovary and conceptus on the uterine natural killer cells during early pregnancy.

Comparison of organochlorine pesticides and PCB residues among hawksbill (Eretmochelys imbricata) and green (Chelonia mydas) turtles in the Yucatan Peninsula and their maternal transfer.

Characterization of a subtropical hawksbill sea turtle (Eretmocheyles imbricata) assemblage utilizing shallow water natural and artificial habitats in the Florida Keys.

Trace elements distribution in hawksbill turtle (Eretmochelys imbricata) and green turtle (Chelonia mydas) tissues on the northern coast of Bahia, Brazil.