differentiation factors in ovary of the Indian wall lizard (Hemidactylus flaviviridis) with emphasis on differential expression and gonadotropic regulation of bmp15 and gdf9

Accepted Manuscript Repertoire of bone morphogenetic proteins and growth/differentiation factors in ovary of the Indian wall lizard (Hemidactylus flaviviridis) with emphasis on differential expression and gonadotropic regulation of bmp15 and gdf9 Mamta Tripathy, Manisha Priyam, Umesh Rai PII: DOI: Reference:

S0016-6480(17)30368-4 http://dx.doi.org/10.1016/j.ygcen.2017.08.015 YGCEN 12731

To appear in:

General and Comparative Endocrinology

Received Date: Revised Date: Accepted Date:

9 May 2017 28 July 2017 14 August 2017

Please cite this article as: Tripathy, M., Priyam, M., Rai, U., Repertoire of bone morphogenetic proteins and growth/ differentiation factors in ovary of the Indian wall lizard (Hemidactylus flaviviridis) with emphasis on differential expression and gonadotropic regulation of bmp15 and gdf9, General and Comparative Endocrinology (2017), doi: http://dx.doi.org/10.1016/j.ygcen.2017.08.015

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Repertoire of bone morphogenetic proteins and growth/differentiation factors in ovary of the Indian wall lizard (Hemidactylus flaviviridis) with emphasis on differential expression and gonadotropic regulation of bmp15 and gdf9

Mamta Tripathy1, Manisha Priyam1, Umesh Rai1* 1

Comparative Immunoendocrinology Laboratory, Department of Zoology,

University of Delhi, Delhi 110007, India *Corresponding Author: Department of Zoology, University of Delhi, Delhi 110007, India; [email protected]

Abstract Analysis of ovarian transcriptome of Indian wall lizard demonstrates the existence of several bone morphogenetic proteins (bmp1, 2, 3, 3b, 7, 8, 15) and growth/differentiation factors (gdf5, 9) for the first time in reptilian ovary. The characterization of putative fulllength/partial protein sequences of BMPs (BMP2, 3, 3b, 7, 15) and GDF9 showed high homology of their TGF-β domain with that of other vertebrates while BMP1 bore homology to zinc-dependent metalloprotease. Phylogenetic analyses showed clustering of BMPs and GDF9 from wall lizards with that of squamates lying in close proximity to chelonia, crocodilia and aves. This study also correlates the expression of ovarian bmp15 and gdf9 with folliculogenesis. Level of bmp15 dramatically increased with the onset of follicular growth in early recrudescence and attained peak during late recrudescence whereas gdf9 sharply decreased during recrudescence as compared to regression. Nonetheless, expression of these growth factors decreased appreciably with the formation of vitellogenic follicle during breeding phase. Ovarian expression of bmp15 and gdf9 appeared to be regulated by gonadotropin as bmp15 considerably increased while gdf9 decreased in parallel to follicular development after administration of 3 injections of FSH. Expression of both the growth factors declined with the prolongation of treatment that led to formation of early/late vitellogenic follicle. Our in vitro study revealed stimulatory effect of FSH on expression of bmp15 and gdf9 in early growing, previtellogenic and early vitellogenic follicles. In light of in vitro results, FSH-induced in vivo decline in gene expression seems to be due to some other FSH-induced factor. Keywords: Bone morphogenetic factors, growth/differentiation factors, reptile, wall lizard, temporal expression pattern, gonadotropic regulation

1. Introduction The follicular growth and development in ovary is regulated by a complex interplay of extra- and intraovarian factors. In mammals, follicular development until preantral stage remains independent of gonadotropins and is regulated by intraovarian factors, while gonadotropins are indispensable for the development of antral and preovulatory follicles (Rajkovic et al., 2006). Among numerous intraovarian factors, members of transforming growth factor β (TGF-β) superfamily are reported to play key role in regulation of ovarian functions (Knight and Glister, 2006; Otsuka et al., 2011, Rossi et al., 2016). They are secreted as preproproteins comprising of an N-terminal signal peptide followed by a proregion and a C-terminal mature domain. The functional form of these proteins is homo- or heterodimer linked by intermolecular disulphide bond or noncovalent interaction (Chang et al., 2002, Juengel and McNatty, 2005, Peng et al., 2013). Based on structural features, members of the TGF-β superfamily are classified into five subfamilies such as TGF-β subfamily, bone morphogenetic protein (BMP) subfamily, growth/differentiation factor (GDF) subfamily, activin/inhibin subfamily and glial cellderived neurotropic factor subfamily (Knight and Glister, 2006). The physiological significance of BMPs and GDFs in recruitment of ovarian follicles (Lee et al., 2001; Nilsson and Skinner, 2003), proliferation of follicular cells (Otsuka et al., 2000; Vitt et al., 2000a,b; Otsuka and Shimasaki, 2002; Kedem et al., 2011), regulation of gonadotropin receptor expression (Otsuka et al., 2001c), steroidogenesis (Elvin et al., 2000; Otsuka et al., 2001a, b; Souza et al., 2002; Pierre et al., 2004; Brankin et al., 2005; Glister et al., 2004; Pierre et al., 2005; Kedem et al., 2011), oocyte maturation (Kathirvel et al., 2013; Li et al., 2014) and cumulus expansion (Elvin et al., 1999; 2000; Kathirvel et al., 2013; Peng et al., 2013) has been extensively

studied in mammals. However, studies on involvement of BMPs and GDFs in control of folliculogenesis in non-mammalian vertebrates are limited to a few fishes like Danio rerio (Clelland et al., 2006, 2007; Liu and Ge, 2007; Clelland and Kelly, 2011), Dicentrarchus labrax (Halm et al., 2008; García-López et al., 2011), Oncorhynchus mykiss (Lankford and Weber, 2010), Carassius auratus gibelio (Liu et al., 2012) and bird Gallus domesticus (Ongabesan et al., 2003; Johanson et al., 2005; Elis et al., 2007). These growth factors have been shown to regulate granulosa cells proliferation (Ongabesan et al., 2003; Johanson et al., 2005), ovarian steroidogenesis (Ongabesan et al., 2003; Elis et al., 2007), expression of tight junction proteins (Clelland and Kelly, 2011) and oocyte maturation (Clelland et al., 2006, 2007; Peng et al., 2009) in fishes and hen. Surprisingly, no such effort has been made in reptiles despite the fact that they are phylogenetically important being ancestor to aves and mammals. In view of this, current study was undertaken to demonstrate the existence of several members of BMP and GDF subfamily in ovary of the Indian wall lizard, Hemidactylus flaviviridis. In addition, we investigated the expression pattern of ovarian bmp15 and gdf9 and its correlation with follicular development during different reproductive phases of wall lizards as these two growth factors have been reported to be obligatory for growth of ovarian follicles in mammals (Dong et al., 1996; Carabatsos et al., 1998; Galloway et al., 2000; Yan et al., 2001; Juengel et al., 2002; Hanrahan et al., 2004; Wang et al., 2013). Also, efforts were made to understand the gonadotropic regulation of ovarian bmp15 and gdf9 in wall lizards. 2. Materials and methods 2.1. Animals

Adult female Indian wall lizards, Hemidactylus flaviviridis (body weight 8-10 g) were procured locally, maintained in wooden cages (6’ × 1.5’ × 1.5’) with wire mesh on top and sides, and fed live houseflies ad libitum. Wall lizards were acclimated to laboratory conditions (12L:12D light regimen at room temperature) for a week prior to experimentation or sample collection. Detailed experimental protocols were approved by the Institutional Animal Ethics Committee, Department of Zoology, University of Delhi, Delhi, India. Guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) were followed for maintenance and sacrifice of lizards. One side ovary of the sacrificed lizards was used for total RNA extraction while other side was processed for routine histology. Six micron thick ovarian sections were stained with Harris’ hematoxylin and eosin. Images were captured by Nikon Eclipse 90i micropscope using NIS elements AR software. 2.2. Reagents and culture media Dulbecco’s modified Eagle’s medium (DMEM) and TRI reagent were purchased from Sigma-Aldrich Chemicals Co. (St. Louis, MO, USA). Ovine follicle stimulating hormone (FSH) was procured from National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK, CA, USA). Other chemicals that were used in the present study are RevertAid first strand cDNA synthesis kit (Thermo Scientific, Waltham, MA, USA), SYBR green (Applied Biosystems, CA, USA), DNAse and DNAse buffer (Fermentas, Waltham, MA, USA), Wizard SV Gel and PCR Clean-Up System (Promega, Wisconsin, USA), Taq DNA polymerase and buffer as well as dNTP (Bangalore Genei Pvt. Ltd., Bengaluru, India). The culture medium was supplemented with 40 µg/ml gentamycin, 100 µg/ml streptomycin, 100 IU/ml penicillin (Ranbaxy India Ltd, New Delhi, India) and pH was adjusted to 7.2. Prior to use, heat-inactivated charcoal-striped fetal calf serum

(FCS; Biological Industries, Kibbutz Beit Haemek, Israel) was supplemented in the medium. 2.3. Sequence characterization Transcripts for several members of BMP and GDF subfamily were identified from annotated ovarian transcriptome sequencing data of H. flaviviridis (Bioproject I.D. PRJNA324371) and verified using nucleotide BLAST (Basic Local Alignment Search Tool). Accession number of individual sequences submitted to NCBI is enlisted in Table 1. Open reading frames (ORF) of the sequences were predicted using NCBI ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) and Expasy Translate Tool (http://web.expasy.org/translate/) was used to predict amino acid sequences. Predicted full-length proteins or partial proteins having complete mature domain of BMPs (BMP1, 2, 3, 3b, 7, 15) and GDF (GDF9) were selected for further analyses. The sites for signal peptide cleavage, phosphorylation and N-glycosylation were predicted using SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/), NetPhos 2.0 (http://www.cbs.dtu.dk/services/NetPhos/) and NetNGlyc 1.0 (http://www.cbs.dtu.dk/service+s/NetNGlyc/), respectively. Simple Modular Architecture Research Tool (SMART; http://smart.embl-heidelberg.de/) was used for domain prediction. 2.4. Phylogenetic analyses Selected BMPs (BMP1, 2, 3, 3b, 7, 15) and GDF9 were subjected to phylogenetic analysis using Mega 6.0 (Tamura et al., 2013). Briefly, protein sequences of wall lizard were aligned with that of reptiles and representative of other vertebrate classes (Accession numbers listed in Supplementary data 1) using MUSCLE (Multiple Sequence Comparison by Log-Expectation) alignment tool (Mega 6.0 package). The alignment files generated for BMPs and GDF9 were used to construct maximum-likelihood trees for each

of the selected growth factors. Also, a common tree was constructed to analyze interrelationship between various BMPs and GDF9 of the wall lizard. 2.5. Temporal expression of ovarian bmp15 and gdf9 Among members of BMP and GDF subfamily, bmp15 and gdf9 are reported essential for ovarian follicular development in mammals (Otsuka et al., 2011). Therefore, expression of these two growth factors was studied in the lizard ovary during different reproductive phases. The ovarian cycle of Indian wall lizard has been delineated broadly into 3 phases, regressed extending from May to July, recrudescent from August to February and breeding from March to April (Sanyal and Prasad, 1967; Guraya, 1976). Since recrudescent phase extends for a considerably long duration when extensive folliculogenesis from development of non-growing follicles to formation of large previtellogenic follicle occurs, we subdivided this phase into early recrudescence (August-October) and late recrudescence (November-February). Based on ovarian cycle, female lizards were sacrificed during mid of each reproductive phase: regressed (June), early recrudescent (September), late recrudescent (December) and breeding phase (late March). One side ovary of each lizard was frozen in liquid nitrogen and stored at -80 oC until total RNA extraction. Ovary of 5 animals was pooled to make one sample for RNA extraction. 2.6. Gonadotropic regulation of ovarian bmp15 and gdf9 expression 2.6.1. In vivo study The wall lizards H. flaviviridis were procured in the month of August (onset of recrudescence). After acclimation, they were administered FSH intraperitoneally (30 µg FSH in 100 μl saline/injection/lizard) on alternate day. Animals were sacrificed 48 h after 3rd, 7th and 11th injection. The experimental design including dose and duration of FSH treatment as well as saline-injected control group was determined based on earlier study in wall lizards (Rai and

Haider, 1989) in which control lizards receiving saline injection for different duration maximally upto 3 weeks did not show any difference in their ovarian weight and follicular diameter. Therefore, in the present study, animals receiving 11 injections of comparable volume of vehicle were considered as control. One side ovary of different experimental groups including control was dissected out and used for histological analysis while other side was immediately frozen in liquid nitrogen and stored at -80 oC until further use. For gene expression analysis, three samples (one side ovary of 3 lizards per sample) were prepared for each experimental and control group. 2.6.2. In vitro study To understand the stage-specific direct role of gonadotropin in regulation of bmp15 and gdf9, follicles of different stages (early growing, previtellogenic and early vitellogenic) were separated out from both side ovaries of 30 lizards during breeding phase. For each stage of follicle (early growing, previtellogenic and early vitellogenic), 6 experimental sets, 10 follicles per experimental set, were made. They were incubated in complete culture medium containing 5% heat-inactivated charcoal-stripped FCS for 24 h. Thereafter, medium was discarded and 6 experimental sets for each stage of follicles were divided into two groups, each consisted of 3 sets of 10 follicles/set (N=3 for each experimental group). In one group, follicles were incubated in complete culture medium containing 0.5 µg/ml FSH for 6 h. The second group was kept as control where follicles were incubated in complete culture medium without FSH for the same duration. After incubation, follicles of each set from both groups for early growing, previtellogenic and early vitellogenic stage were collected separately, frozen in liquid nitrogen and stored at 80 oC until RNA extraction. Thus, 3 different sets, each set consisting of 10 follicles, were used to analyze the gene expression in control and similarly in FSH-treated group for specific stage of follicles. The concentration and duration of FSH treatment were

decided based on earlier in vitro study in wall lizards (Khan and Rai, 2004) and our pilot experiment. The culture was maintained at 25 oC in humidified incubator with 5% CO2. 2.7. Total RNA extraction and cDNA preparation Total RNA was extracted from tissue samples following TRI reagent protocol. RNA was quantified at 260/280 absorbance (Nanodrop spectrophotometer, ND 1000, Nanodrop technologies, USA) and its integrity was analysed on bioanalyser (Agilent technologies, California, USA). Samples having absorbance ratio (A260/280) ranging from 1.8 to 2.0 and RIN (RNA Integrity Number) greater than 5 were processed for cDNA preparation. Further, 1 µg RNA was treated with DNase to remove any genomic DNA contamination which was then reverse transcribed using RevertAid first strand cDNA synthesis kit following the manufacturers’ protocol. 2.8. Quantitative real time PCR (qPCR) Absolute quantification of ovarian bmp15 and gdf9 was carried out during different reproductive phases to examine the correlation between growth factors and follicular development. For gonadotropic regulation, expression of bmp15 and gdf9 was assessed in ovary of FSH-injected and vehicle-injected lizards. Also, level of these growth factors was determined in ovarian follicles of different stages incubated with or without FSH. Prior to absolute quantification, transcripts of bmp15 and gdf9 obtained from ovarian transcriptome sequencing data were validated by partial sequencing. Briefly, primers for bmp15 (5’ CCCTGAGGTACATGCTG 3’ and 3’ GCAATCATACCCTCATACC 5’) and gdf9 (5’ CCCTGGATCGTGTTACTGC 3’ and 3’ GTCACATCAATTTCAACCC 5’) were designed and PCR product was commercially sequenced. The partial sequences were aligned against their specific transcripts using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/). For absolute quantification of gene expression, specific primers were designed for bmp15 (5’ CAGCCACGTCCTCCATGT 3’ and 3’ CAGCCTGGTCCAAAGTCC 5’) as well as gdf9 (5’

GACCGCAACCAGACTCC 3’ and 3’ CGTGGAGTGAAGAGCCG 5’) and their optimum annealing temperatures were standardized. The amplification efficiency of each primer set (105.1% for bmp15 and 100.5% for gdf9) was tested using serial dilutions of cDNA and melt curve analysis was used to confirm amplification of a single product. The qPCR reactions were performed using cycle steps as follows: pre-incubation at 95°C for 10 min followed by 39 cycles of amplification that included denaturation at 95°C for 10 sec, annealing and extension at gene-specific annealing temperature for 1 min. PCR amplicons obtained using qPCR primers for bmp15 and gdf9 were eluted and their serial dilutions were used as standards in respective qPCR reactions. Log concentrations of the standards were plotted against mean Cq values to construct standard curves. Further, gene-specific standard curve was used to extrapolate the concentrations of bmp15 and gdf9 in each sample. All samples were run in triplicate and no template control (NTC) was run with all the reactions. 2.9. Statistical analysis The variation in ovarian expression of bmp15 and gdf9 along the reproductive cycle and under in vivo effect of FSH was analyzed by one-way analysis of variance (ANOVA) followed by Newman–Keuls multiple range test (P < 0.05). Student’s t-test (P < 0.05) was used to examine the in vitro effect of FSH on early growing, previtellogenic and early vitellogenic follicles as compared to their respective controls. Data are presented as mean ± standard error of mean. 3. Results 3.1. Sequence Characterization De novo ovarian transcriptome sequencing data of wall lizards revealed the presence of several members of BMP and GDF subfamilies such as bmp1, 2, 3, 3b, 7, 8, 15 and gdf5, 9. Transcripts of bmp1, 2, 15 and gdf9 possessed a complete coding sequence (CDS)

encoding full-length proteins with signal peptide, proregion and TGF-β domain (Fig 1A, B, F, G, respectively). Although transcripts of bmp3, 3b and 7 contained partial CDS, they were adequate to encode complete mature region (TGF-β domain) of their respective proteins (Fig 1C-E). The truncated transcripts of bmp8 and gdf5 were not suitable for sequence characterization. The TGF-β domain of BMP2, 3 and 7 contained seven cysteine residues (Fig 1B, C, E) while six cysteine residues were present in that of BMP3b, 15 and GDF9 (Fig 1D, F, G). Unlike other BMPs and GDF9, BMP1 lacked TGF-β domain and in lieu of this a zinc dependent metalloprotease domain (ZnMc domain) followed by five conserved CUB domains and two EGF-CA (EGF-like calcium binding) domains were predicted in BMP1 of wall lizards following SMART analysis (Fig 1A). In addition, bioinformatic analyses of transcripts of BMP1, 2, 3, 3b, 7, 15 and GDF9 showed the presence of several phosphorylation and N-glycosylation sites. 3.2. Phylogenetic analysis Phylogenetic trees for BMP1, 2, 3, 3b, 7, 15 and GDF9 showed clustering of these ovarian growth factors from wall lizard H. flaviviridis with respective orthologs of other squamates (Fig. 2A-G). Further, growth factors of squamates were phylogenetically close to crocodilia, chelonia and aves, except squamate BMP1 that exhibited proximity to its mammalian ortholog. Among reptiles, growth factors (except BMP15 and GDF9) from crocodiles were positioned closer to aves. Overall, BMPs and GDF9 from Sauropsids comprising of reptiles and birds were evolutionarily closer to mammals, except BMP3 that showed proximity to amphibian ortholog. A separate evolutionary tree constructed for examining interrelationship between various growth factors of wall lizards exhibited the presence of two clades, one consisting of BMP1 and 7 and other consisting of BMP2, 3, 3b, 15 and GDF9 (Fig. 2H). Further, a close phylogenetic relationship was observed between BMP3 and BMP3b. Similarly, BMP15 was found to be closer to GDF9.

3.3. Histological changes in ovary during different reproductive phases During quiescent phase, ovary (~1mg weight) was regressed and consisted of a germinal bed, stromal connective tissue having tiny oocytes either naked or surrounded by single layer of flattened pre-granulosa cells, and small extrastromal follicles (Fig 3A). In early recrudescence, ovarian weight increased (3-4 mg weight) due to rise in number and growth of extrastromal follicles of different sizes arranged in hierarchy. A complete loss of stromal connective tissue was observed. Granulosa cells were differentiated into three types, small apical/basal, round intermediate and large pyriform cells (Fig 3B). The development of thecal layer was noticed in large-sized follicles. Further growth of ovarian follicles during late recrudescent phase led to a marked increase in ovarian weight (9-12 mg weight). In the largest follicle, thecal layer was highly developed and polymorphic granulosa cells tend to become monomorphic thus, resulting in a decrease of thickness of granulosa layer (Fig 3C). During breeding phase, deposition of yolk granules in the ooplasm of the largest follicle caused manyfold increase in ovarian weight. In vitellogenic follicle, granulosa cells were monomorphic and arranged in a single layer. The homogenous zona pellucida became striated and referred as zona radiata (Fig 3D). 3.4. Expression of bmp15 and gdf9 during different reproductive phases The ovarian expression of bmp15 and gdf9 showed a marked (ANOVA, P < 0.05) variation depending on reproductive phases of the wall lizard (Fig. 4A, B, respectively). Transcript level of bmp15 increased significantly (P < 0.05) in early recrudescent as compared to that of regressed phase. A further rise in its expression was observed from early to late recrudescence. However, expression of bmp15 sharply declined (P < 0.05) during breeding phase. In contrast to bmp15, highest expression of gdf9 was recorded during regressed phase. Its expression significantly (P < 0.05) declined with ovarian follicular development during recrudescent and breeding phase.

3.5. Histological changes in ovary after FSH administration Ovary of control lizards at the onset of recrudescence consisted of 2-3 small follicles, from non-growing to early growing stage. A remnant of stromal connective tissue was present. Polymorphic differentiation of granulosa cells into small, intermediate and pyriform was less evident in these follicles (Fig 5A). The differentiation of theca was conspicuously absent (Fig 5A). Administration of FSH induced the ovarian follicular growth and differentiation. Remnants of stromal connective tissue observed in control ovaries (Fig 5A) disappeared, number of ovarian follicles increased and development of thecal layer commenced in large-sized follicles after 3 injections of FSH (Fig 5B). Seven injections of FSH caused further growth of oocytes. The polymorphic granulosa cells tend to become monomorphic and thecal layer was highly developed in the largest follicle (Fig 5C). The presence of early stage of vitellogenic follicle with monomorphic granulosa and highly developed theca was observed in a few lizards after 7 injections (Fig 5D). A large preovulatory follicle containing yolk granules in ooplasm was observed in ovary of all lizards receiving 11 injections of FSH. The homogenous zona pellucida of growing follicles became striated in vitellogenic follicles (Fig 5E). 3.6. Effect of FSH on bmp15 and gdf9 expression 3.6.1. In vivo study Expression of bmp15 in ovary of wall lizards increased significantly (P < 0.05) after 3 injections of FSH when compared to control (Fig. 6A). However, a steep (P < 0.05) decline in ovarian bmp15 expression was observed after 7/11 injections of FSH. In case of gdf9, an insignificant decrease (16-28%) in its expression in lizard ovary was observed after treatment with FSH as compared to control (Fig. 6B). 3.6.2. In vitro study

Incubation of different stages of ovarian follicles (early growing, previtellogenic and early vitellogenic follicles) with FSH for 6 h showed a marked (P < 0.05) increase in expression of both, bmp15 and gdf9, in all the three stages of follicles (Fig. 7A, B, respectively) as compared to their respective controls. 4. Discussion The existence of various members of BMP and GDF subfamilies such as BMP1, 2, 3, 3b, 4, 5, 6, 7, 8, 15 and GDF9, 15 has been evidenced in the ovary of mammals (Knight and Glister, 2006; Staff et al., 2010). With regard to their expression in the ovary of nonmammalian vertebrates, BMP2a, 2b, 4, 6, 7a, 15, 16 and GDF9 have been reported in fishes (Liu and Ge, 2007; Halm et al., 2008; Peng et al., 2009, Feiner et al., 2009; Lankford and Weber, 2010; Li and Ge, 2011; Luckenbach et al., 2011) and BMP2, 4, 6, 7, 15 and GDF9 in birds (Onagbesan et al., 2003; Johanson et al., 2005; Elis et al., 2007). The present study in wall lizards for the first time documents the existence of several BMPs and GDFs in the ovary of a reptile. Ovarian transcriptome of wall lizards revealed the presence of bmp1, 2, 3, 3b, 7, 8, 15 and gdf5, 9. However, bmp4, 5, 6 that have been demonstrated to play critical role in regulation of follicular development and steroidogenesis in mammals (Rossi et al., 2016), were absent from ovarian transcriptomic data of wall lizards. BMP4 has been shown to stimulate growth of primordial follicles (Nilsson and Skinner, 2003; Ding et al., 2013) while BMP5 and 6 are reported to be critical for development of secondary follicles in several mammalian species (Pierre et al., 2005; Shimizu et al., 2004; Glister et al., 2004; Frota et al., 2011). Moreover, these growth factors have been shown to modulate ovarian progesterone production (Shimasaki et al., 2004; Pierre et al., 2005; Kayani et al., 2009), survival of oocytes (Ding et al., 2013) and follicular lutenization (Shimasaki et al., 2004). Interestingly, our study showed

the presence of gdf5 in lizard ovary which has not been reported so far in the ovary of any other vertebrate. The predicted mature domain/TGF-β domain of ovarian BMP2, 3, 3B, 7, 15 and GDF9 of wall lizards showed high homology with their mammalian orthologs. Like mammals (Knight and Glister, 2006), 7 cysteine residues were observed in TGF-β domain of BMP2, 3 and 7 of wall lizards. In mammals, one of these cysteine residues is implicated in stabilizing the dimer by forming an intermolecular disulphide bond (Griffith et al., 1996) while other 6 residues of each monomer form a cysteine knot consisting of 3 intramolecular disulphide bonds (McDonald and Henderickson, 1993). In view of this, we speculate that these cysteine residues might be involved in stabilizing monomer as well as dimer of BMP2, 3 and 7 of wall lizards. In case of mammalian BMP15 and GDF9, noncovalent interaction has been suggested for dimer stabilization (Liao et al., 2003) since they lack fourth cysteine residue involved in intermolecular disulphide bonding between monomers. A similar non-covalent interaction between monomers might be suggested for BMP3b, BMP15 and GDF9 of wall lizards as they have only 6 cysteine residues and lack the fourth one. Phylogenetic analyses of these growth factors showed an evolutionary divergence for BMPs and GDF9 of squamates from that of other reptiles, as reptilian BMPs and GDF9 clustered in two subclades, one consisting of squamates and other comprising of chelonians and crocodilians. Further, the crocodilian sequences were found to be closer to that of aves which is in consistence with other studies where a close evolutionary relationship between these two groups has been documented (Janke and Amason, 1997; Organ et al., 2008). BMP15/GDF9 and BMP3/3b have been shown to be paralogous (Katoh and Katoh, 2006; Monestier et al., 2014) and this is corroborated by our study in which BMP15 formed a clade with GDF9 while BMP3 with BMP3b. During analysis of phylogenetic interrelationship between various growth factors of wall lizards,

BMP15 clustered with GDF9 while BMP3 with BMP3b. Our results are in agreement with other studies in which BMP15/GDF9 and BMP3/3b have been shown to be paralogous (Katoh and Katoh, 2006; Monestier et al., 2014). The ovarian growth factors BMP15 (Yan et al., 2001) and GDF9 (Dong et al., 1996; Carabatsos et al., 1998) have been reported obligatory for folliculogenesis in mammals (Knight and Glister, 2006). Homozygous point mutations in bmp15 (Galloway et al., 2000; Hanrahan et al., 2004) and gdf9 (Hanrahan et al., 2004) have been shown to cause infertility in ewes. The critical role of these ovarian growth factors in regulating fertility is also reported in rodents. Female gdf9 null mice are reported infertile since ovarian follicular development is arrested at the stage of primary follicle (Dong et al., 1996; Carabatsos et al., 1998) and bmp15 / female mice are subfertile (Yan et al., 2001) due to decreased ovulation rate. Studies in rat have implicated GDF9 (Vitt et al., 2000a, b; Hayashi et al., 1999; Nilsson and Skinner, 2002) and BMP15 (Otsuka et al., 2000) in proliferation of granulosa cells and development of follicles from primary to small preantral stage. Moreover, expression of bmp15 in the ovary of rat has been reported to increase from primary to secondary follicle stage and then remain unchanged until ovulation (Erickson and Shimasaki, 2003). In agreement, bmp15 expression in ovary of wall lizards showed a considerable increase from regressed to early recrudescence when growth and development of ovarian follicles commenced. The high level of bmp15 remained unaltered until late recrudescence during which follicles undergo further growth and development. However, our study revealed an appreciable decrease in ovarian bmp15 expression during breeding phase when ovary possessed predominantly vitellogenic preovulatory follicle. Our observation gains support from a study in European seabass wherein high expression of bmp15 during previtellogenesis has been shown to decline at the time of vitellogenesis (Halm et al., 2008). A different expression pattern, contrary to bmp15, was observed for gdf9 until formation of large

previtellogenic follicles in ovary of wall lizards. The level of gdf9 that was highest in regressed ovary considerably declined with the growth and development of follicles during early/late recrudescence, indicating that gdf9 might be involved in formation of germinal bed, primordial follicles and tiny extrastromal follicles having multi-layered granulosa. Nonetheless, in parallel to bmp15, expression of gdf9 further decreased in lizard ovary of breeding phase. A similar expression pattern of gdf9 has been demonstrated in ovarian follicles of hen (Johnson et. al., 2005; Elis et. al., 2007), rainbow trout (Lankford and Weber, 2010), gibel carp (Liu et al., 2012) and zebrafish (Liu and Ge, 2007) wherein highest level of expression was noted in the smallest primary stage follicles while low level was observed in follicles of advanced stages. It is worth mentioning that expression profile of ovarian gdf9 and bmp15 has not been investigated along the reproductive cycle in reptiles and hence, based on our observation in the Indian wall lizards we speculate that these growth factors might not be importantly involved beyond previtellogenic stage in reptiles. With regard to gonadotropic regulation of ovarian bmp15 and gdf9, studies are scanty and present confusing picture. Guéripel et al. (2006) have reported that FSH administration in immature mice stimulates bmp15 expression in total ovaries as well as cumulus-oocyte complexes (COCs) isolated from the largest follicles while Thomas et al. (2005) have shown the inhibitory effect of FSH on bmp15 expression in COCs of immature mice. Unlike bmp15, expression of gdf9 is reported to remain unaffected by FSH in total ovary (Guéripel et al., 2006) and in COCs (Guéripel et al., 2006; Thomas et al., 2005). With regard to non-mammalian vertebrates, reports are limited and confined to fishes. Treatment with hCG which is widely used to induce ovarian follicular development and ovulation in fishes, has been reported to increase bmp15 expression in ovarian follicles of gibel carp (Chen et al., 2012). In case of gdf9, hCG is shown to inhibit

the expression in ovarian fragment from zebrafish in a concentration-dependent manner (Liu and Ge, 2007). Further, incubation of different stages of zebrafish ovarian follicles, previtellogenic, early vitellogenic and fully grown vitellogenic, with hCG showed inhibition of gdf9 expression only in fully grown vitellogenic follicles (Liu and Ge, 2007). On the contrary, hCG is reported to stimulate gdf9 expression in fully grown follicles of gibel carp (Liu et al., 2012). However, in an in vitro study in coho salmon Oncorhynchus kisutch, FSH has been reported ineffective in inducing gdf9 expression in mid-late cortical alveolus stage ovarian follicles. The current in vivo and in vitro study in wall lizards, for the first time in reptiles, evidenced the involvement of gondaotropin in regulation of ovarian growth factors (bmp15 and gdf9) and their correlation with growth and development of the ovary. Ovarian expression of bmp15 increased dramatically in parallel to follicular development after 3 injections of FSH. Interestingly, expression of bmp15 in total ovary declined concomitantly with the development of early/late vitellogenic follicle in lizards receiving 7/11 FSH injections. Overall, the pattern of bmp15 expression and histological changes in lizard ovary in response to exogenous FSH were comparable to that observed during recrudescent and breeding phases. Our in vitro experiment, however, showed stimulatory effect of FSH on bmp15 expression in all the stages of follicles, suggesting that decrease in bmp15 expression in total ovary during breeding phase or after long term FSH treatment might be due to some other inhibitory factor(s) produced in the in vivo system. In case of gdf9, the significant decline in ovarian expression observed with the development of ovarian follicles during early recrudescence continued until breeding phase of wall lizards. Interestingly, ovarian gdf9 expression did not decline significantly after administration of FSH that stimulated the ovarian growth and development. On the contrary to in vivo results, incubation of early growing, previtellogenic and early vitellogenic follicles with FSH showed considerable

upregulation of gdf9 expression in all stages of follicles. Taken together, we speculate that inhibition of gdf9 in the in vivo system could be due to other factor(s) that would have overridden the stimulatory effect of FSH and eventually inhibited the gdf9 expression. In conclusion, a large repertoire of bmps and gdfs is present in the ovary of the Indian wall lizard. Members of BMP and GDF subfamilies of TGF-β superfamily expressed in lizard ovary bear high homology with their counterparts in other vertebrates. The expression profile of bmp15 and gdf9 along the reproductive cycle and in FSHtreated lizards implicates their involvement in folliculogenesis up to previtellogenic stage. Nonetheless, in vitro observations established the direct upregulatory role of FSH in ovarian expression of bmp15 and gdf9 in wall lizards. 5. Declaration of interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. 6. Funding This work was supported by Research and Development grant from the University of Delhi, Delhi and Purse grant from Department of Science and Technology, Government of India. The first author is thankful to the University Grants Commission, New Delhi for senior research fellowship. 7. Acknowledgement We are thankful to Prof. Namita Agrawal, Department of Zoology, University of Delhi, India for extending her microscopy and imaging facility for the histological analysis in the current study.

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Figure legends Fig. 1. Amino acid sequences of wall lizard BMPs and GDF9. (A) Predicted sequence of BMP1. The zinc-dependent metalloprotease (ZnMc) domain is enclosed in square brackets ( ) while the calcium-binding EGF-like (EGF_CA) domain is enclosed with parentheses ( ). The CUB domains have been shaded gray. (B-G) Predicted protein sequences of BMP2, 3, 3b, 7, 15 and GDF9. The conserved cysteine residue is marked by closed triangle ( ) and sites where the cysteine is substituted is represented by closed star ( ). The RXXR motif that are putative site for propeptide cleavage have been enclosed in parentheses ( ) and the mature protein has been shaded gray. For all the protein sequences A-G site for signal peptide cleavage is marked by a closed arrow ( ). Probable sites for phosphorylation have been underlined while those for N-glycosylation are enclosed in open rectangles ( ). Fig. 2. (A-G) Phylogenetic tree denoting evolutionary position of BMPs and GDF9 from Indian wall lizard H. flaviviridis (A: BMP1, B: BMP2, C: BMP3, D: BMP3b, E: BMP7, F: BMP15, G: GDF9). Multiple sequence alignment for each growth factor was constructed using respective protein sequences from representatives of all vertebrate classes (accession number of sequences enlisted in supplementary data 1). Further, these alignments were used to construct maximum likelihood trees based on Jones-TaylorThornton (JTT) model. The numbers at nodes indicate bootstrap values in percentage. (H) Phylogenetic tree showing interrelationship between lizard BMP1, 2, 3, 3b, 7, 15, and GDF9. BMPs and GDF9 of wall lizard were aligned to construct maximum likelihood tree based on Jones-Taylor-Thornton (JTT) model. The numbers at nodes indicate bootstrap values in percentage. Fig. 3. Histological changes in the ovary of Indian wall lizard Hemidactylus flaviviridis along its reproductive cycle. (A) Section of regressed ovary showing presence of germinal bed comprising of oogonia, stromal connective tissue containing a few primordial follicles in which oocytes are surrounded by a single layer of flattened pregranulosa cells and a portion of small extrastromal follicle. (B) Section of lizard ovary of early recrudescent phase. Note the presence of multilayered polymorphic granulosa consisting of pyriform, intermediate, small apical and small basal cells, poorly developed thecal layer and homogenous zona pellucida. (C) Showing two adjacent follicles from ovary of late recrudescent phase. Granulosa cells are polymorphic in one follicle, while they are losing their polymorphic character and tend to become monomorphic in another follicle. Thecal layer and zona pellucida are relatively more developed. (D) Section of

fully vitellogenic follicle from lizard’s ovary of breeding phase. Note the presence of single-layered monomorphic granulosa, highly developed theca, striated zona radiata and extensive deposition of yolk granules in ooplasma. (GB- Germinal bed, G- Granulosa layer, O- oogonia SCT- Stromal connective tissue, EF-Early growing follicle, PrFPrimordial follicle, OP- Ooplasma, IM- Intermediate cell, PF- Pyriform cells, AP- Apical cells, B- Basal cells, T- Theca, ZP- Zona pellucida, ZR- Zona radiata, Y- Yolk) (magnification 400X).

Fig. 4. Temporal expression of (A) bmp15 and (B) gdf9 in the ovary of wall lizards H. flaviviridis. Three samples (N=3) were used to run qPCR in each phase. Each sample consisted of one side ovary of 5 wall lizards. Data were analyzed by one-way analysis of variance (ANOVA) followed by Newman-Keuls multiple range test (P < 0.05). Means ± SEM with same superscript (a-b/a-c) do not differ significantly.

Fig. 5. Histological changes in ovary of wall lizards after treatment with FSH for different durations. (A) Section of ovary of vehicle-injected control lizard showing remnants of stromal connective tissue and a portion of early growing follicles. Thecal layer is less evident and granulosa cells are poorly differentiated into pyriform, intermediate, small apical and small basal cells. (B) Section showing ovary of lizard receiving 3 injections of FSH on alternate day. Mark the presence of polymorphic granulosa, relatively developed theca and homogenous zona pellucida. Granulosa cells are fully differentiated into pyriform, intermediate, small apical and small basal cells. (C, D) Section of ovary of lizards receiving 7 injections of FSH on alternate day. Early vitellogenic (C) or late vitellogenic (D) follicles were observed in ovaries of FSH-treated lizards. Polymorphic granulosa tending to become monomorphic, well-developed theca and striating zona pellucida could be seen in early vitellogenic follicles (C). Late vitellogenic follicle is characterized by presence of large yolk granules, monomorphic granulosa, striated zona pellucida and highly developed theca (D). (E) Showing a portion of fully developed vitellogenic follicle from ovary of wall lizard receiving 11 injections of FSH on alternate day. Thickness of monomorphic granulosa is relatively reduced. Note the deposition of relatively large-sized yolk droplets in ooplasm. (G- Granulosa layer, SCT- Stromal connective tissue, OPOoplasma, IM- Intermediate cell, PF- Pyriform cells, AP- Apical cells, B- Basal cells, TTheca, ZP- Zona pellucida, ZR- Zona radiata, Y- Yolk) (magnification 400X).

Fig. 6. In vivo effect of ovine follicle stimulating hormone (FSH) on expression of (A) bmp15 and (B) gdf9 in ovary of wall lizards receiving 3, 7, 11 injections of FSH (30 µg FSH/injection/lizard, intraperitoneally on alternate day). Three samples (N=3), each comprised of one side ovary from 3 wall lizards, were run for each treated group and control receiving comparable volume of vehicle for maximum duration. Data were analysed by one-way analysis of variance (ANOVA) followed by Newman-Keuls multiple range test and represented as mean ± SEM (different superscripts ( a-b) differ significantly, P < 0.05).

Fig. 7. In vitro effect of FSH on expression of (A) bmp15 and (B) gdf9 in follicles of different stages, early growing, previtellogenic, early vitellogenic, collected from lizard ovaries of breeding phase. For each stage of follicle, 6 experimental sets, 10 follicles/set, were made. After incubating in complete culture medium for 24 h, 6 experimental sets for each stage were divided into 2 groups (3 sets of ten follicles/set/group; N=3 per experimental group). In one group, follicles were incubated with FSH for 6 h while second group was kept as control (without FSH). After incubation, follicles of each set/experimental group were processed for expression analysis. Student t-test (P<0.05) was performed to compare the unpaired means (control vs FSH-treated) for each stage of follicles. Data represented as mean ± SEM. Significant difference between control and respective experimental group is indicated by an asterisk (*). Table Titles Table 1

NCBI accession numbers for BMP1, 2, 3, 3b, 7, 8, 15 and GDF5 and 9 of wall lizard S. No.

Gene Name 1.

Accession Number Bone

KX555535

morphogenetic protein 1 2.

Bone

KX555536

morphogenetic protein 2 3.

Bone

KX555537

morphogenetic protein 3 4.

Bone

KX555538

morphogenetic protein 3b 5.

Bone morphogenetic protein 7

KX555539

6.

Bone

KX579491

morphogenetic protein 8 7.

Bone

KX555540

morphogenetic protein 15 8.

Growth

KX579490

differentiation factor 5 9.

Growth differentiation factor 9

KX555541

A. (BMP1) 10 20 30 40 50 60 MPASRGGLLF LSFLVLGQAM DFTDYSYVIE EEEEDTDPID YKDPCKAAAF SGDIALDAED 70 80 90 100 110 120 LKYFQVDRVV DLTRHTIERS VTNSSGNSSN NTSIRPRRQQ RRRKLDRGRS RSRRAATSRP 130 140 150 160 170 180 ERVWPDGVIP YVISGNFSGS QRAIFRQAMR HWEKHTCVTF LERTDEDSYI VFTYRPCGCC 190 200 210 220 230 240 SYVGRRGGGP QAISIGKNCD KFGIVVHELG HVIGFWHEHT RPDRDAHVSI IRENIQPGQE 250 260 270 280 290 300 YNFLKMEPEE VESLGETYDF DSIMHYARNT FSKGIFLDTI LPKYNVNGMQ PSIGQRTRLS 310 320 330 340 350 360 KGDIAQARKL YRCPACGETL QDSQGNFSSP EFPNGYSAHM HCIWRISVTP GEKIRLNFTS 370 380 390 400 410 420 LDLYRSRLCW YDYLEVRDGF WRKAALRGRF CGNKLPESIL SSDSRLWIEF RSSSNWVGKG

D. (BMP3b) 10 20 30 40 50 60 MKRSRQFGEL LLCAELDSGK KYHDISEEGA SHWPYILVYA NDLAISEPNS LALSLQRYDP 70 80 90 100 110 120 FPSAESPLLN VSSNTRTKRG AHLSMPVQNN ELPGLVDNSN KHNEQDLWRS AYRSLKSKAS 130 140 150 160 170 180 QRARRRKEQG NVEILPKSQV LNFDEKTMRE AQQKQWSEPA VCSRRYMKVD FADIGWSEWV 190 200 210 220 230 240 ISPKSFDAYY CAGGCEFPMP KIVRPSNHAT IQSIVKAVGI IPGIPEPSCV PNTMNSLSVL 250 260 FLDQSDNIVL KVYPNMSVDS CACR

E. (BMP7)

430 440 450 460 470 480 FFAVYEAICG GDMKKDSGHI QSPNYPDDYR PNKACVWKIT VTEGFHVGVS FQAFEIERHD

10 20 30 40 50 60 MLDLYNAMSV EEEGAGGGGE ADGYSYPYKP LFSTQGPPLA SLQDSNFLTE ADMVMSFVNL

490 500 510 520 530 540 SCAYDYLEVR DGNSETSNLI GRYCGYDKPD DIKSTSNKLW MKFVSDGSIN KAGFAVSFFK

70 80 90 100 110 120 VEHDREFYHQ RSHRREFRFD LSRIPEGEAV TAAEFRIYKD YIRERFDNET FQIRVYQVLQ

550 560 570 580 590 600 EVDECSRPNN GGCEQRCVNT LGSYKCACDP GYELAPDRRR CEAACGGFLT KLNGSITSPG

130 140 150 160 170 180 EHPGRDSDLF LLDSRTIWAA EEGWLVFDIT ATSNDWVLNP QHNLGLQLSV DSIDGQSINP

610 620 630 640 650 660 WPKEYPPNKN CIWQLVAPTQ YRISLQFDFF ETEGNDVCKY DFVEVRSGLT AEAKLHGKFC

190 200 210 220 230 240 KLAGLIGRHG PQNKQPFTVV FFKATEVHLR SIRSAGGKPR NQKSKAPKNQ EAFRVSNVGE

670 680 690 700 710 720 GAEKPDVITS QYNNMRIEFK SDNTVSKKGF KAHFFSDKDE CSKDNGGCQH ECLNTFGSYE

250 260 270 280 290 300 NSSSDQRQAC KKHELYVSFR DLGWQDWIIA PEGYAAYYCE GECAFPLNSY MNATNHAIVQ

730 740 750 760 770 780 CQCRSGFVLH DNKHDCKEAG CDHKMTSISA TITSPNWPDK YPNKKECTWA ITTTPGHRVK

310 320 330 340 350 TLVHFINPDT VPKPCCAPTQ LNAISVLYFD DSSNVILKKY RNMVVRACGC H

790 800 810 820 830 840 LIIRELDIEG HQECTYDHLE VYNGKDAKAP VLGRFCGTKE PDPIISSSNR MFLRFFSDNS 850 860 870 880 890 900 VQKKGFEASY TSECGGQMRA EVKTKDLYSH AQFGDNNYPG GSDCEWVIVA EEGYGVELIF 910 920 930 940 950 960 QTFEIEEEAD CGYDYMELFD GYDGTAPRLG RYCGSGPPEE VYSAGDSVMV RFHSDDTINK 970 980 KGFHLRYTST KFQDTLHTRK

B. (BMP2)

F. (BMP15) 10 20 30 40 50 60 MANLDALCLC GALFLLTVLF PLEYGVGVGQ VEAASVGAVS GAPSLPLIQV LLGKGPPKAR 70 80 90 100 110 120 PQGRNRRLVR GQPLRYMLNL YRSVADRHGR PRRNRKLATN TVRLVKPFAK ARQPGAGPWL 130 140 150 160 170 180 VWNLDYHLEI QPQVEHLVRA TLVYSQTLSL AQGQFLCTAE LLSGKDAVPR TTLSLTSPRV 190 200 210 220 230 240 GKRNATIFSA KDSWVEMDLS MYLQPWVWTA QNSHVLHVRH ACAYVGQPWG SILDPSWEEA

10 20 30 40 50 60 MIAGTHSLLA LLLYQVLLGG SASLIPEVGD RRRFSGDLIR AAPLQLSEGI LREFELRLLN

250 260 270 280 290 300 VSLNDPFLLL YLNDTLNGLW TRLGGLGSGE VPFEEKHLRR PVRSRQARQA GSLALDLPSY

70 80 90 100 110 120 MFGLKRRPTP SKNAVIPPYM LELYRLHVSQ KPSPMDYNLE RATSRANTVR SFHHEETLEE

310 320 330 340 350 360 LRTNSIKKRE CSLHPFRVSF QQLGWDHWII APHSYQPQYC KGNCPRILHF GYHSPNHAII

130 140 150 160 170 180 LPERSGRTSR RFFFNLTSIP SGEYITSAEL QVFRQHLADA FETNSSSYHR INIYEIIKMA

370 380 390 400 410 QNFINELVDK SVPPPSCVPY EYSPISILMM EQNGTILYKV YEDMIAKSCT CR

190 200 210 220 230 240 SEASQEPVTR LLDTRLVHHN ASKWETFDVT PAIMRWIAHG QPNHGFVIEV VHLDDESSVS 250 260 270 280 290 300 KRHVRISRSL HQDDASWSLV RPLLVTFGHD GKGQPLHKRE KRQAKHKQRK RLKSNCKRHP 310 320 330 340 350 360 LYVDFNDVGW NDWIVAPPGY SAFYCHGECP FPLADHLNST NHAIVQTLVN SVNSKIPKAC 370 380 390 CVPTELSAIS MLYLDENEKV VLKNYQDMVV EGCGCR

G. (GDF9) 10 20 30 40 50 60 MKILWGILTC CCIGLFFSGV LCSPNSRDEG GSQPLLVSDR EAAEPLSMLL FPPDAKSSHA 70 80 90 100 110 120 VLSPLFKVLT GHSHQEENDG GQRPQPDSRA LSYMKRLYKM YATKEGIPKA NKSHLYNTVR 130 140 150 160 170 180 LFTPRAECKH PAEGQVKGDL HSVDLLFNLD CVTALEHLFK SVLLYSLDKS VSKSSAITCT 190 200 210 220 230 240 CNLIVKEHDP SNQVCSSVPH TMTVELKRRW VEIDVTSFLQ PLIASHKRSI HIAVNFTCLK

C. (BMP3) 10 20 30 40 50 60 MAPVGRHSWK RVHSTYEPYI LVYANDSAIS EPESVVSSLQ GHWTPLPRGF SKLDSHSKAD 70 80 90 100 110 120 RGERREKRSA SILLPLQNNE LPGAEYQFSE DEGWEERKHY KTLQPRLAER AKSKKKQRKN 130 140 150 160 170 180 NHQKSQTLQF DEQTLKKARR KQWNEPRYCA RRYLKVDFAD IGWSEWIISP KSFDAYYCSG 190 200 210 220 230 240 ECQFPIPKAL KPSNHATIQS IVRAVGVVSG IPEPCCVPDK MSSLSILFFD ENKNVVLKVY 250 PNMTVESCAC R

250 260 270 280 290 300 DNEPLDSKWD FFNMALVSPS LLLYLNDTSE QAYHRRDMNP IWSDLKNSPL MNPSEKTGKE 310 320 330 340 350 360 PIQDERASRH RRDEDGKVKN TSAPPYNLSQ FYQQFLFPQN ECELQNFWLK FSQLKWDRWI 370 380 390 400 410 420 IAPHGYNPHY CKGECPRVVG HRYGSPVHTM VQNIIYEKLD SSVPKPSCVP AEYSPLSILK 430 440 LESDRSVAYK EYENMIATKC TCR

A.

C.

B.

D.

F. E.

G.

H.

A.

B.

GB

ZP

T

O B

SCT AP

G

B

C.

IM OP

PrF

EF

PF

D. ZP

PF

T

ZR G T

AP

OP

IM

Y

bmp15 mRNA level (pg/mg tissue wt.)

b

B.

4000 3500

b

3000 2500

a

2000 1500

a

1000 500

gdf9 m RNA level (pg/mg tissue wt.)

4500

A.

0

20

a

18 16 14 12 10 8

b

b

6 4 c

2 0

Regressed

Early Recrudescent

Late Recrudescent

Breeding

ZP

B.

A. SCT

B

OP IM

T

G PF AP

G

OP

C.

D. ZP OP Y

ZR G G

T Y

E.

T T G G

ZRZR

Y Y

T

250

2500

A.

a

b 2000

1500

a

1000 a

a

500

gdf9 mRNA level (pg/mg tissue wt.)

bmp15 mRNA level (pg/mg tissue wt.)

B. 200

a 150

100

50

0

0

Control

3 Injections

7 Injections

11 Injections

a

a

1800 1600 1400

*

1200 *

1000 800 600 400 200 0

18 16

Previtellogenic follicles

Early vitellogenic follicles

Control

500

*

14 12

400

10

*

8 6

300 200

4 100 2 0

Early growing follicles

600

*

Early growing follicles

Experimental

Previtellogenic follicles

0

Early vitellogenic follicles

gdf9 mRNA level in follicles incubated with FSH (pg/mg tissue wt.)

(B) * gdf9 mRNA level in control follicles (pg/mg tissue wt.)

bmp15 mRNA level (pg/mg tissue wt.)

(A) 2000

Highlights: 1. First documentation of the repertoire of BMPs and GDFs in the ovary of a reptile 2. First report on temporal expression of bmp15 and gdf9 in ovary of a reptile 3. First evidence that FSH directly regulates bmp15, gdf9 in non-mammalian vertebrate