Luteal cell steroidogenesis in relation to delayed embryonic development in the Indian short-nosed fruit bat, Cynopterus sphinx

Luteal cell steroidogenesis in relation to delayed embryonic development in the Indian short-nosed fruit bat, Cynopterus sphinx

ARTICLE IN PRESS ZOOLOGY Zoology 112 (2009) 151–159 www.elsevier.de/zool Luteal cell steroidogenesis in relation to delayed embryonic development in...

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ZOOLOGY Zoology 112 (2009) 151–159 www.elsevier.de/zool

Luteal cell steroidogenesis in relation to delayed embryonic development in the Indian short-nosed fruit bat, Cynopterus sphinx Karukayil J. Meenakumari, Arnab Banerjee, Amitabh Krishna Department of Zoology, Banaras Hindu University, Varanasi 221 005, Uttar Pradesh, India Received 24 January 2008; received in revised form 8 April 2008; accepted 24 April 2008

Abstract The primary aim of this study was to determine the possible cause of slow or delayed embryonic development in Cynopterus sphinx by investigating morphological and steroidogenic changes in the corpus luteum (CL) and circulating hormone concentrations during two pregnancies of a year. This species showed delayed post-implantational embryonic development during gastrulation of the first pregnancy. Morphological features of the CL showed normal luteinization during both pregnancies. The CL did not change significantly in luteal cell size during the delay period of the first pregnancy as compared with the second pregnancy. The circulating progesterone and 17b-estradiol concentrations were significantly lower during the period of delayed embryonic development as compared with the same stage of embryonic development during the second pregnancy. We also showed a marked decline in the activity of 3b-hydroxysteroid dehydrogenase, P450 side chain cleavage enzyme, and steroidogenic acute regulatory peptide in the CL during the delay period. This may cause low circulating progesterone and estradiol synthesis and consequently delay embryonic development. What causes the decrease in steroidogenic factors in the CL during the period of delayed development in C. sphinx is under investigation. r 2009 Elsevier GmbH. All rights reserved. Keywords: Chiroptera; Corpus luteum; Embryonic development; Reproductive delay; Steroidogenic factors

Introduction Four different types of reproductive delay have been shown in female bats: delayed ovulation, delayed fertilization, delayed implantation and delayed development (post-implantation). Delayed ovulation and delayed fertilization are uncommon in mammals but are common in temperate-zone vespertilionid bats (Oxberry, 1979; Racey, 1982; Krishna, 1999), and have also been observed in several subtropical and tropical bats (Krutzsch, 1979; Krishna, 1999). Delayed implantation Corresponding author. Tel.: + 919451938967.

E-mail address: [email protected] (A. Krishna). 0944-2006/$ - see front matter r 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.zool.2008.04.007

is the most common form and has been reported in several mammals belonging to eight different orders including Chiroptera (Mead, 1993). Among Chiroptera, delayed implantation has been described in the bentwinged bats, Miniopterus schreibersii (Bernard and Bojarski, 1994) and in the equatorial fruit bat, Eidolon helvum (Mutere, 1967). Post-implantation delay in development is the least common type of reproductive delay in mammals and has only been demonstrated in bats (Heideman and Powell, 1998; Krishna, 1999). Although delayed embryonic development is now described in many bat species, the cause and control of delayed development has not yet been extensively investigated. In most mammals that exhibit delayed

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implantation the steroidogenic activity of the corpus luteum (CL) is suppressed and plasma progesterone concentrations are low during the delay (skunk: Sinha and Mead, 1976; roe deer: Sempere, 1977; badger: Bonnin et al., 1978; mink: Martinet et al., 1981; Sundquist et al., 1989; long-fingered bat: Kimura et al., 1987; Crichton et al., 1989; Bernard and Bojarski, 1994). The mechanism of delayed development has been studied extensively in Macrotus californicus; Crichton et al. (1990) showed low circulating levels of progesterone during the delayed period. Whether the low progesterone level during the period of delayed embryonic development is due to abnormal luteal cell development, low luteotrophic hormone stimulation, or suppression of the luteal steroidogenic enzyme activities has not yet been investigated. To provide additional information on the causes of developmental delay, we studied the short-nosed fruit bat, Cynopterus sphinx, which breeds twice in quick succession at Varanasi, India (Krishna and Dominic, 1983). The bat is unusual in that its gestation length varies significantly in the two successive pregnancies of the year. The first pregnancy is initiated between late October and early November and lasts for about 150 days, whereas the second (summer) pregnancy is initiated in April and lasts for about 125 days (Krishna and Dominic, 1983). The prolonged gestation period during the first pregnancy of C. sphinx is due to delayed or slowed embryonic development during November to December (Meenakumari and Krishna, 2005). The developmental arrest occurs at the gastrula stage of the embryo (Meenakumari and Krishna, 2005) during November to December. This is in contrast to the summer pregnancy, in which no arrest is observed and development proceeds relatively faster (Meenakumari and Krishna, 2005). The aim of the present study was to evaluate the steroidogenic capacity of the ovaries (particularly the Table 1.

CL) during the two pregnancies of the year to define their role for delayed embryonic development. Specifically, the present study investigated: (a) morphological changes in the CL, (b) circulating steroid (progesterone and estradiol) and luteinizing hormone (LH) concentrations, (c) the changes in 3b-hydroxysteroid dehydrogenase (3b-HSD), side chain cleavage enzyme (SCC) and steroidogenic acute regulatory protein (StAR) activity in the ovary during the two pregnancies in C. sphinx.

Materials and methods Animals All experiments were conducted in accordance with the principles and procedures approved by Banaras Hindu University, Departmental Research Committee. The female bats (C. sphinx) utilized in this study were captured alive on and around Banaras Hindu university premises, Varanasi, India, between the years 1998 and 2004. Captured animals were immediately transported to the laboratory. Body mass of bats was recorded as soon as they were brought to the laboratory (within 2 h of capture). Females weighing 43 g or more and having a wing span exceeding 46 cm were classified as sexually mature (Krishna and Dominic, 1983). Female bats were classified into the following reproductive phases (Krishna and Dominic, 1983): quiescence, recrudescence, preovulatory, first pregnancy, post-partum oestrus and second pregnancy (Table 1).

Histological procedures For histological procedures 30 female bats were euthanized between 15.00 and 16.00 h by decapitation, the blood was collected and their reproductive tracts

Classification of reproductive stages of Cynopterus sphinx.

Quiescence Recrudescence

August to early September Mid-September to early October

Pre-ovulatory and ovulatory

Mid-October to midNovember

First pregnancy (winter pregnancy) Post-partum oestrus

Late October to March Late March to early April

Second pregnancy (summer pregnancy)

Early April to July

Reproductively inactive phase, ovary lacks antral follicle and corpus luteum; pregnant females not found during this period. Beginning of reproductive activity, ovaries contained both antral and pre-antral follicles; corpus luteum not present in the ovary; pregnant females not found during this period. Ovary contained one large pre-ovulatory follicle; newly formed corpus luteum in the ovary first noticed by October and ovary of some females contain Graafian follicle until early November. Ovary contained well-developed corpus luteum ipsilateral to the pregnant side; antral follicles are present in the ovary contralateral to the pregnant side. Ovary contralateral to the parturient side contained preovulatory follicle. The simultaneous occurrence of pregnant and lactating females during this period indicates the incidence of a post-partum oestrus. Ovary contained well developed corpus luteum ipsilateral to the pregnant side.

Based on the reproductive cycle of C. sphinx at Varanasi, India (Krishna and Dominic, 1983).

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were immediately dissected out. Serum was separated out by centrifugation within 1 h and stored at 20 1C until assayed. The ovary and uterus were fixed in Bouin’s fluid for 24 h, dehydrated through grades of ethyl alcohol, cleared in xylene and embedded in paraffin wax. Most of the ovaries were serially sectioned transversely at a thickness of 6–7 mm. The histological sections were stained with hematoxylin and eosin.

Steroid assays The blood collected after decapitation of female bats was centrifuged at 3000 rpm for 20 min at 4 1C and then serum was collected which was used for steroid assays. Progesterone assay We used a radioimmunoassay (RIA) kit for progesterone from ICN Biomedicals Inc., Costa Mesa, CA, USA. First, 25 ml of each standard and sample were added to the anti-progesterone coated tubes. Progesterone I125 (1 ml) was then added to each tube. The tubes were incubated at 37 1C for 60 min. After incubation the tubes were decanted and empty tubes were checked in a gamma counter (Beckman, Geneva, Switzerland). The concentration of progesterone in the samples was deduced by extrapolation from the standard curve. All the tubes for progesterone were assayed together and intra-assay variation was less than 10%. Steroid assays for bat samples have been previously validated (Abhilasha and Krishna, 1996). Estradiol assay For 17ß-estradiol we used a RIA kit from Orion Diagnostica, Finland. To the anti-estradiol coated tubes 100 ml each of standards, controls and bat serum samples were added. Diluted estradiol (500 ml) tracer (labeled with I125) was added to each tube and following gentle mixing incubated for 2 h at 37 1C. After incubation, each tube was decanted, washed in 1 ml of washing solution and the count per minute (cpm) was measured in a Beckman gamma counter. Concentration of estradiol in the samples was deduced by extrapolation from the standard curve. All the tubes for estradiol were assayed together and intra-assay variation was less than 10%. Steroid assays for bat samples have been previously validated (Abhilasha and Krishna, 1996). LH assay The circulating LH concentration in the bats was measured using an RIA kit (RIAK-10 for humans) obtained from the Board of Radiation and Isotope Technology (BRIT), Mumbai, India. We validated this LH assay by diluting pooled serum samples from bats with assay buffer and testing the series against a standard curve. Dilutions of bat serum ran parallel to

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the standard curve, indicating the suitability of the assay for use in bats. Duplicate 0.1 ml serum samples were transferred in 12  75 mm2 polystyrene tubes. Assay buffer (0.2 ml) was added to each sample followed by 0.1 ml human LH antiserum. To each tube LH I125 (0.1 ml) was added. The tubes were vortexed and incubated at room temperature for 4 h. Anti-rabbit IgG (0.1 ml) and 1.0 ml of polyethylene glycol were added simultaneously to all the tubes except to the total count tubes, vortexed and kept at room temperature for 20 min. Tubes were then centrifuged at 1500g for 20 min. After centrifugation the supernatants were discarded and precipitates were counted in a gamma scintillation counter (Beckman, Geneva, Switzerland). Standards (5.0–100 mIU/ml), zero standard and blank tubes were also processed with the samples. All the samples were run in one assay. The intra-assay variation was less than 10%.

3b-hydroxysteroid dehydrogenase activity For the 3b-HSD assay another 20 female bats were euthanized by decapitation. Ovaries containing a CL were dissected out and stored at 70 1C until 3b-HSD activity was determined according to the method described by Tanaka et al. (1993, with slight modifications). Ovaries were weighed and homogenized in 0.25 mol l1 sucrose at 0 1C with a glass–glass homogenizer at a concentration of 20 mg wet weight ml1. The homogenates were centrifuged at 10,000g for 30 min. The supernatant fluids were used as the enzyme solution. The assay mixture, which consisted of 40 mmol l1 glycine–NaOH (pH 9.4), 0.9 mg bovine serum albumin (BSA), 0.5 mmol l1 oxidized nicotinamide adenine dinucleotide (NAD +) and 50 ml of enzyme was preincubated at 37 1C for 5 min. The reaction was started by the addition of substrate solution containing pregnenolone (0.1 mg ml1 in ethanol) in a final volume of 800 ml. The activity was determined spectrophotometrically by measuring the absorbance of NADH at 340 nm. One unit of enzyme activity was defined as the amount of enzyme producing 1 mmol l1 NADH. The values were expressed as mU mg1 protein. The method of Lowry et al. (1951) was used for protein determination, with BSA used as the standard.

Immunoblot Six ovaries containing a CL were pooled to produce a 10% tissue homogenate in homogenization buffer containing 20 mmol l1 Tris HCl (pH 7.4), 1 mmol l1EDTA, 1 mmol l1 phenylmethylsulfonyl fluoride, 25 mg/ml aprotinin, 0.1% (wt/vol) SDS. Protein extraction and immunoblot were performed as described previously

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(Chanda et al., 2004). Equal amounts of protein (determined according to Lowry et al., 1951) were loaded onto an SDS-PAGE gel (10%) for electrophoresis. Thereafter, proteins were transferred electrophorectically to nitrocellulose membranes (Sigma-Aldrich, St. Louis, USA) overnight at 4 1C. Nitrocellulose membranes were blocked for 60 min with Tris-buffered saline (TBS; 50 mmol l1 Tris (pH 7.5), 150 mmol l1 NaCl) containing 5% fat-free dry milk and incubated with rabbit antihuman StAR antibody (at a dilution of 1:500) and rabbit anti-human P450 SCC (at a dilution of 1:600) for 1 h at room temperature. The primary antibody was detected with anti-rabbit IgG antibody coupled to horseradish peroxidase, using an enhanced chemiluminescence (ECL) detection system. Experiments were repeated thrice with the same results. Equal loading was confirmed with Ponceau S staining.

Statistical analysis All data are presented as means7standard error of the mean (SEM). The significance of the differences in steroid concentrations and in enzyme activities between groups was determined by one way analysis of variance (ANOVA) followed by Duncan’s multiple range test or a t-test. The data were considered significant if Po0.05.

Fig. 1. Seasonal variations in the diameter of (A) corpus luteum, (B) luteal cell and luteal cell nucleus of Cynopterus sphinx.

Results Morphological features of the corpus luteum Starting in October, the ovary of C. sphinx females contained a single spherically shaped corpus luteum (CL) ipsilateral to the reproductive tract carrying the newly ovulated ovum or conceptus. The CL contained a single type of oval to polygonal luteal cells, vascular elements, connective tissue and fibroblasts. Soon after ovulation, luteal cells were loosely arranged in the CL. The newly formed CL contained a large central part filled with undifferentiated stromal cells and surrounded by a layer of flat stromal cells. Strands of connective tissue divided the CL into broad strands of cells. From ovulation to the blastula stage, the CL gradually increased in size and became highly vascularized. The CL reached its maximum diameter of about 1.02770.047 mm shortly after implantation of the blastocyst. The fully formed CL appeared as a solid structure without a central cavity and completely embedded in the ovarian stroma. During early pregnancy the CL had large luteal cells with prominent nuclei and clearly defined boundaries. At mid-pregnancy, the CL did not change in size and occupied a major part of the ovarian stroma. The luteal

cells though decreased in size as compared with the early pregnancy, still appeared turgid, with clearly defined boundaries and with vesicular nuclei containing prominent nucleoli. Small vacuoles were visible in the cytoplasm of the luteal cells. By late pregnancy, signs of luteal regression became apparent. Several luteal cells appeared shrunken, with ill-defined boundaries and with increased density of fibroblast cells. The regressed CL persisted until parturition. CL diameter, luteal cell diameter and nuclear diameter during the two successive pregnancies of the year are shown in Fig. 1A and B. Differences in the morphological features and duration of CL were apparent during the two successive pregnancies of the year. During the first (winter) pregnancy (Fig. 2A–D), the CL of females with embryos in delay (during November and December) contained large luteal cells with vesicular nuclei. Examination of the uterine horn at the site of implantation showed a lack of placenta formation during the delayed period. Soon after the end of delayed development in early January the CL contained large luteal cells. In most females with a uterine swelling of about 4–8 mm, large luteal cells were still abundant, but their nuclei significantly decreased in size (Po0.05;

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Fig. 2. Histology of the corpus luteum during different stages of pregnancy in Cynopterus sphinx. (A–D) First pregnancy; (E–G) second pregnancy. Magnification: 250  . (A) Corpus luteum soon after ovulation in November showing well differentiated luteal cells with well defined boundaries and vesicular nuclei. (B) Corpus luteum during delayed embryonic development in December showing large polygonal luteal cells with defined boundaries and vesicular nuclei without any signs of regression. (C) Corpus luteum during mid-pregnancy in January showing a decline in luteal cell size (arrow) and shrunken nuclei. (D) Corpus luteum during late pregnancy in February showing regressive luteal cells (arrow). (E) Corpus luteum during early stages of the second pregnancy in April showing well differentiated luteal cells with large and vesicular nuclei. (F) Corpus luteum during mid-pregnancy in May showing large polygonal luteal cells with well defined cell boundaries and vesicular nuclei. (G) Corpus luteum during late pregnancy in June showing decline in luteal cell size (arrow) and shrunken nuclei.

Fig. 1B). Females with a uterine swelling greater than 4 mm showed a well developed placenta. The CL showed signs of regression by the end of January or early February. During the second (summer) pregnancy (Fig. 2E–G), the CL quickly attained the maximum size by the time of implantation and remained large and active till midpregnancy. Most females with a uterine swelling of 15–17 mm in early June had CLs with signs of regression. Duration of CL activity was found to be

much longer during the first pregnancy as compared with the second pregnancy of the year.

Changes in serum progesterone and 17b-estradiol concentration Changes in circulating progesterone and 17b-estradiol concentrations during the two successive pregnancies of C. sphinx are shown in Figs. 3 and 4.

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Fig. 3. Monthly changes in the circulating progesterone level of Cynopterus sphinx. O-1 ¼ October, pre-ovulatory; O-2 ¼ October, post-ovulatory; M-1 ¼ March, pre-ovulatory; M-2 ¼ March, post-ovulatory.

The assay results showed two peaks of serum progesterone concentrations corresponding with the two pregnancies in C. sphinx, with a relatively lower (Po0.05) circulating progesterone concentration during the first pregnancy as compared with the second pregnancy of the year (Fig. 3). During the first pregnancy, the circulating progesterone level increased at the time of ovulation in October, but subsequently remained unchanged until December. The circulating progesterone concentration then increased steeply from December onwards to attain a peak during March. The progesterone concentration decreased significantly (Po0.05) at the end of the first pregnancy in March. During the second pregnancy the circulating progesterone level increased steeply (Po0.05) from the beginning in April and attained a second peak during June to July. A plateau of the circulating progesterone level as observed during the first pregnancy (November to December) was not observed during the second pregnancy. The circulating estradiol concentration showed two short peaks during the first pregnancy and one large peak during the second pregnancy (Fig. 4). Similar to the progesterone concentration, the serum estradiol concentration was also low during the delay period of

Fig. 4. Monthly changes in the circulating estradiol level of Cynopterus sphinx.

Fig. 5. Changes in the circulating LH level of Cynopterus sphinx during different reproductive phases. Pre-o ¼ preovulatory; Preg I ¼ first pregnancy; Post-p ¼ post-partum oestrus; Preg II ¼ second pregnancy; Qui ¼ quiescence.

the first pregnancy. It increased again to attain a short peak immediately after the delay period in January and another short peak corresponding with the post-partum oestrus in March. During the second pregnancy, serum estradiol remained high during May and June.

Changes in circulating LH concentrations Changes in the circulating LH concentration during the reproductive cycle of C. sphinx are shown in Fig. 5. The results showed two peaks of serum LH concentration corresponding with the two periods of ovulation in C. sphinx. The serum LH level remained high during both pregnancies. There was no indication of a decline in LH level during the period of delayed development (P40.05).

Biochemical estimation of 3b-HSD activity in the CL The 3b-HSD activity in the ovaries containing a CL during different stages of two pregnancies is shown in Fig. 6. The enzyme activity was moderately high during

Fig. 6. Monthly changes in the 3b-hydroxysteroid dehydrogenase (3b-HSD) activity in Cynopterus sphinx ovaries containing a corpus luteum.

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Fig. 7. Immunoblot analysis of P450 SCC enzyme in ovaries of Cynopterus sphinx containing a corpus luteum during the early phase of two successive pregnancies (April and November to December). Two immunoreactive bands of SCC were noticed at 49 and 42 kDa during both pregnancies. A nonspecific band at 42 kDa was found in the immunoblot for P450 SCC enzyme.

early pregnancy in November, declined significantly (Po0.05) during the period of delayed development in December and increased significantly in January following the delay period. During the second pregnancy, it remained moderately high in April and May.

SCC activity SCC activity in the ovary with CL was studied using immunoblot followed by densitometry during the early phase of the two successive pregnancies (November to December and April) and results are described in Fig. 7. Two immunoreactive bands of SCC were noticed at 49 and 42 kDa during the period of delayed development in November to December as well as during the early phase of the summer pregnancy in April. The band intensity was significantly low (Po0.05) during the winter pregnancy in November to December.

StAR activity StAR activity in the ovary containing a CL was studied by immunoblot followed by densitometry (Fig. 8). During the embryonic development the StAR protein showed a single immunoreactive band of varying intensity at 30 kDa. The results further showed a marked decline (Po0.05) in StAR activity during the delayed period in November to December as compared with the early phase of the second pregnancy.

Discussion The aim of the present study was to compare the changes in CL and circulating hormone concentrations

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Fig. 8. Immunoblot analysis of StAR protein in ovaries of Cynopterus sphinx containing a corpus luteum during the early phase of two successive pregnancies (April and November to December). StAR protein showed a single immunoreactive band at 30 kDa, with a markedly declined activity during the delayed period as compared with the early phase of the second pregnancy.

during the two pregnancies of the year in C. sphinx in order to find out the possible cause of slow or delayed embryonic development. The investigation showed that CL developed rapidly following ovulation during both pregnancies of the year. A significant increase (Po0.05) in circulating progesterone level at the time of ovulation supports this finding. The study showed a decline (Po0.05) in the activity of the steroidogenic factors in the CL together with low circulating progesterone and 17b-estradiol concentrations during the period of delayed development of the first (winter) pregnancy as compared with the second (summer) pregnancy. This suggests that slow embryonic development during the gastrulation stage of the first pregnancy might be due to reduced synthesis of progesterone and estradiol. This is the first study demonstrating a slow post-implantational (gastrulation) process due to decreased synthesis of progesterone and estradiol. It is well known that a low progesterone level causes delayed implantation in several mammalian species (Wade-Smith et al., 1980; Bernard et al., 1991). Comparison of morphological features of the CL during the two periods of pregnancy in C. sphinx suggests normal luteinization during both pregnancies. The luteal cell size showed no significant changes during delayed development of the first pregnancy. In contrast, Fleming (1971) showed smaller luteal cells during the period of delayed development in Artibeus jamaicensis. Bernard et al. (1991) observed a decrease in CL volume during embryonic diapauses in the Natal clinging bat (Miniopterus schreibersii natalensis). The present study is thus in agreement with earlier findings in the roe deer Capreolus capreolus, which also has an active CL and low circulating progesterone level during developmental delay (Aitken, 1981).

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The present study in C. sphinx showed a significantly low circulating concentration of progesterone and estrogen during the period of delayed development during the first pregnancy as compared with the same stage of embryonic development during the second pregnancy. This is in agreement with an earlier study on Macrotus californicus, which also showed low levels of plasma progesterone during the period of delayed development, although the stage of embryonic development during the delayed period is not known in this species (Crichton et al., 1990). In both species the circulating progesterone levels remained low during the developmental delay period and increased significantly at the resumption of normal embryonic development (Crichton et al., 1990). The substantial rise in serum progesterone level coincides with the phase of the most rapid rate of fetal growth. In C. sphinx, the CL continued to produce progesterone during the delay period, though at a low level. This suggests that C. sphinx requires a much higher level of progesterone for post-implantational development. Since this study failed to find a developed placenta during the period of delayed development, it may be hypothesized that the development of the placenta as an additional source of progesterone may be required for post-implantational development in C. sphinx. The delayed embryonic development during the first pregnancy in C. sphinx could be due to the incomplete or delayed placental development. Badwaik and Rasweiler (2001) clearly noted alterations in the differentiation of the placental cytotrophoblast in C. perspillata during the period of delayed embryonic development. What causes the halt in progesterone concentration during delayed embryonic development has not been studied. The present study in C. sphinx showed a decline in the activity of the steroidogenic factors like 3b-HSD, SCC and StAR during the period of delayed embryonic development in the early stage of the first pregnancy as compared with the second pregnancy. This is the first study suggesting that decreased progesterone and estradiol levels found during the period of delayed embryonic development in the early phase of the first pregnancy are due to a significant decline in the activity of the steroidogenic enzymes 3b-HSD, SCC and StAR in the CL of C. sphinx. In a previous study we showed that the suppressed embryonic development in C. sphinx during the winter pregnancy corresponds with the period of fat accumulation (Banerjee et al., 2007). It has also been demonstrated that the circulating leptin level increases during fat accumulation in bats (Srivastava and Krishna, 2007). Interestingly, an increased leptin level associated with suppression in ovarian steroidogenesis is mediated by a decline in the expression of StAR (Salzmann et al., 2004; Srivastava and Krishna, 2007). Thus it appeared that the decrease of StAR in C. sphinx during November to December

might be due to an increased leptin concentration in this bat species during fat accumulation. Leptin directly affects the ovarian function as supported by studies showing the presence of leptin receptor and its mRNA in human, mouse, rat and pig ovaries (Karlsson et al., 1997; Kikuchi et al., 2001; Ruiz-Corte´s et al., 2003). The delayed embryonic development during early winter in C. sphinx may have evolved to synchronize parturition among females and to time parturition appropriately so that birth of the young and lactation may occur during the most favorable season for raising offspring.

Acknowledgements This study was financially supported by the Department of Science and Technology, New Delhi. Antibodies against P450 SCC enzyme and StAR were generously gifted by M.J. Soares (Ralph L. Smith Mental Retardation Research Center, University of Kansas, KS, USA) and D.M. Stocco (Health Sciences Center, Texas Tech University, Lubbock, TX, USA).

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