Molecular Characterization, Tissue Distribution, and Expression Profiling of the CTSD Gene during Goose Ovarian Follicle Development

Cathepsin D (CTSD) is known to be crucial for the degradation and utilization of yolk precursors in ovarian follicles. However, little is known about its expression profiles and physiological actions in avian ovarian cells. In this study, the intact coding sequence of the CTSD gene in geese was cloned for the first time, with a length of 1197 bp. It encoded a polypeptide of 398 amino acids (AA) consisting of a signal peptide and two conserved functional domains (i.e., A1_Propeptide and Cathepsin_D2). The AA sequence of goose CTSD had > 96% similarities with the homologs of turkeys, chickens, and ducks. Results from real-time PCR showed that goose CTSD mRNA was present in all tissues examined, with higher levels in the adrenal gland, liver, heart, and reproductive organs. Furthermore, levels of CTSD mRNA were much higher in goose granulosa layers than in the theca layers in any follicular category. Significantly, its expression remained almost unchanged in the theca layers throughout follicle development, while it increased gradually in the granulosa layers from 2-4 mm to F5 follicles but declined there after. These results suggested that CTSD may regulate goose ovarian follicle development through its actions on both the degradation and absorption of yolk precursors and granulosa cell apoptosis.

In birds, ovarian follicles committed to ovulation are usually arranged in a strict hierarchy, comprising hundreds of visible follicles at different developmental stages (HERNANDEZ & BAHR 2003;HU & ZADWORNY 2017). During the reproductive lifespan of birds, only a very small number of follicles will be ultimately ovulated while most of them will undergo apoptosis which could take place at any developmental stage (JOHNSON et al. 1996;JOHNSON & WOODS 2009). In particular, compared to ducks and chickens with annual egg production numbers of 250, domesticated geese (Anser cygnoides domestica) show poor egg production performance, usually laying 60 eggs per year, which severely impedes the rapid development of the goose industry. Because the fate of ovar-ian follicles determines goose lifetime reproductive performance and depends on a well-organized follicular hierarchy, it is of great theoretic and practical value to identify the factors regulating goose follicle development as well as the underlying mechanisms.
It is known that avian follicle development is not only characterized by the differentiation of follicular cells, including granulosa and theca cells, but also accompanied by the deposition and absorption of liversynthesized yolk precursors, i.e., very low density lipoprotein (VLDL) and vitellogenin (VTG) (ROMANO et al. 2004). Over the past two decades, a lot of studies have been conducted to explore the molecular mechanisms controlling the differentiation of follicular cells (JOHNSON 2015), whereas little is known about the factors regulating the degradation and utilization of yolk precursors as well as their actions in follicular cells. Our previously published studies have suggested that the VLDL receptor (VLDLR) plays a critical role in the transport of yolk precursorsinto goose ovarian follicles and that the VLDLR-mediated endocytosis of lipids as well as endogenously synthesized triglycerides are important for maintaining normal follicle development (HU et al. 2014b;HU et al. 2020). Regarding the reported roles of lysosomal proteinases in lipid degradation (THELEN & ZONCU 2017), it is proposed that they may be responsible for the utilization of yolk precursors in birds. Indeed, cathepsin D (CTSD), a lysosomal aspartyl protease, has been demonstrated to be involved in the degradation and absorption of yolk precursors, thereby providing essential energy and nutrients for oocyte and embryonic development in fish and chickens (RETZEK et al. 1992;KWON et al. 2002;TINGAUD-SEQUEIRA & CERDÀ 2007;PALOMINO et al. 2017). In fish, CTSD mediates the processing of VTG and VLDL, and its derivative yolk proteins and lipids are subsequently utilized for embryonic development (CARNEVALI et al. 1999a). Furthermore, PALOMINO et al. (2017) andCARNEVALI et al. (1999b) have indicated that both the expression level and enzymatic activity of CTSD are key determinants of egg quality in several fish species and could be considered as the molecular markers for egg quality. In addition, CTSD can influence cellular activities, including proliferation, autophagy, and apoptosis, in a range of cell types. It was reported that in human neuroblastoma cells CTSD exerted an antiapoptotic effect and attenuated doxorubicin-induced apoptosis (VITALIA et al. 2008) while in mouse embryonic fibroblasts cytoplasmic CTSD levels regulated the balance between cell survival and cell death (KIM et al. 2013). In the tadpoles of a tropical toad, CTSD was involved in programmed cell death in the epidermis, muscles, spinal cord, and blood cells during tail resorption (MAHAPATRA & MAHAPATRA 2012), and in lepidopteran insects, the differential expression and autophagy-mediated maturation of CTSD determined its roles in cell proliferation and apoptosis, eventually determining the cell fates of tissues during metamorphosis (DI et al. 2020). Thus, we hypothesized that CTSD may influence avian ovarian follicle development not only by mediating the degradation and absorption of yolk precursors but also by regulating follicular cell activities.
The main objectives of the present study were to first clone and characterize the full-length coding sequence (CDS) of the CTSD gene in geese and to subsequently examine its mRNA expression in a wide range of tissues as well as in the granulosa and theca layers of goose follicles at different developmental stages. These data are expected to provide a basis for further clarification of the exact roles of CTSD in avian ovarian follicular cells.

Ethics statement
All experimental procedures involving the manipulation of birds were conducted in concordance with the "Guidelines for Experimental Animals" of the Ministry of Science and Technology (Beijing, China). This study has been reviewed and approved by the Sichuan Agricultural University Animal Ethical and Welfare Committee (Approval No.: 20180034).

Experimental birds and tissue collection
Females from the maternal line of Tianfu meat geese (Anser cygnoide), 35-45 weeks of age and laying in regular sequences of at least 2-3 eggs, were used in all studies described. The geese were kept under natural conditions of light and temperature and were provided with free access to feed and water at the Waterfowl Breeding Experimental Farm of Sichuan Agricultural University (Ya'an campus, Sichuan, China). Among them, three healthy geese having similar body weights were sacrificed by exsanguination, and thereafter, a total of thirteen types of tissue, including heart, liver, spleen, kidney, intestine, muscle, adipose, brain, hypothalamus, pituitary, adrenal gland, ovarian stroma, and oviduct tissues, were collected from each individual. The follicles within each ovary were classified as previously described (HU et al. 2014a;HU et al. 2020) into the prehierarchical 2-4-, 4-6-, 6-8-, and 8-10 mm in diameter follicles as well as the preovulatory F5-F1 (F5<F4<F3<F2<F1 in diameter) follicles. Furthermore, the granulosa layers were separated from the theca layers for all follicular categories according to the method introduced by HU et al. (2014a). All tissue samples were rapidly frozen in liquid nitrogen and finally stored at -80 o C until RNA isolation.

RNA isolation and cDNA synthesis
Total RNA was isolated from each sample using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Its purity and concentration was determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and its integrity was assessed by visualization of the 28S/18S rRNA ratio after electrophoresis on 1.5% agarose gels. The cDNA was then synthesized from 1 µg RNA using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's instructions.

Primer design
According to the reported cDNA sequences of the CTSD gene in chickens, swine, humans, and mice (GenBank accession number: NM_205177.1, NM_001037721.1, NM_001909.4, and NM_009983.2, respectively), three pairs of primers (namely D1-D3) were designed to amplify three overlapping fragments spanning the full-length CDS of the goose CTSD gene. Then, based on the obtained intact CDS, the primer named D4 was designed to examine the relative mRNA expression of the goose CTSD gene in multiple tissues. Meanwhile, the primers named E1, based on the goose ß-ACTIN gene (GenBank accession number: M26111), and E2, based on the goose GAPDH gene (GenBank accession number: MG674174), were also designed and used as internal controls. All of the above primers were designed using Primer Premier 5 software (Primer Biosoft International, Palo Alto, CA, USA)and are listed in Table 1.

RT-PCR
Using the cDNA transcribed from the goose ovary as the template, three overlapping fragments of goose CTSD cDNA were amplified using corresponding primer pairs by reverse transcription (RT)-polymerase chain reaction (PCR). The PCR reactions were performed in a Bio-Rad thermal cycler (Bio-Rad, USA) with the following program: pre-denaturation at 95 o C for 5 min, followed by 35 cycles of denaturation at 95 o C for 30 s, annealing at the primer-specific annealing temperature for 30 s, and extension at 72 o C for 1 min, and a final extension at 72 o C for 10 min. The PCR products were evaluated by DNA electrophoresis on 1.5% agarose gels and purified using a gel extraction kit (Watson Biomedical Inc., Shanghai, China). Target cDNAs were then cloned into the pMD19-T vector (Invitrogen, USA) and sent to Invitrogen Corporation (Applied Invitrogen, Shanghai, China) for sequencing.

Real-time PCR
The mRNA expression levels of the goose CTSD gene in various tissues as well as in the theca and granulosa layers of follicles at different stages of development were measured using real-time PCR. The PCR reactions were performed on the CFX96 ™ Real-Time PCR Detection System (Bio-Rad, USA) using the SYBR Premix Ex Taq ™ II (Takara Biotechnology Co., Ltd., Dalian, China). Reactions were conducted under the following conditions: pre-denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 15 s and annealing/extension at the corresponding temperature of each primer set for 30 s. The no-template controls and negative controls without reverse transcriptase were also included in all qPCR runs. Target specificity for each primer set was validated by melting curve analyses, and the identity of all amplicons was verified by sequencing. All samples were amplified in triplicate and the relative mRNA expression levels of the goose CTSD gene were normalized to the reference genes â-ACTIN and GAPDH using the comparative Cq method (ÄÄCq) (SCHMITTGEN & LIVAK 2008). The primers used for real-time PCR are listed in Table 1. Table 1 Primer pairs used in this study Primer ID Primer name Primer sequence (5' to 3') Real-time PCR GAPDH-R GTGGTGCAAGAGGCATTGCTGAC Note: F, forward primer; R, reverse primer.

Bioinformatics analysis
All amplified fragments of goose CTSD cDNA were assembled using the DNAMAN software. The open reading frame (ORF) Finder tool on the NCBI website was used to predict the ORF of the goose CTSD gene. Homology analyses of both the nucleotide and AA sequences were performed using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) (SIEVERS and HIGGINS 2014). Multiple alignments of the CTSD AA sequences among different species were performed using the DNAMAN software. A phylogenetic tree was constructed using the MEGA 7.0 software. The conserved functional domains and the aspartyl catalytic residues within the AA sequence of the goose CTSD gene were predicted using the CD-search tool on the NCBI website. The putative N-glycosylation site and signal peptide were predicted using the NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/) and SignalP-5.0 Server (http://www.cbs.dtu.dk/services/SignalP/), respectively.

Statistical analysis
Data from the real-time PCR experiments were presented as the mean ±SD of three individuals and were subjected to an analysis of variance (ANOVA) followed by Duncan's Multiple Range Test using SAS 9.4 software (SAS Institute, Cary, USA). P-values below 0.05 were considered statistically significant.

Molecular characterization and sequence analysis of goose CTSD cDNA
Three overlapping fragments were cloned using corresponding primer pairs and assembled to successfully obtain the full-length CDS of the goose CTSD gene (GenBank accession number: MT812700). As shown in Figure 1, the intact CDS of the goose CTSD gene had a length of 1197 bp, which encoded a polypeptide of 398 AA. Results from multiple alignments suggested that there were two active aspartyl residues (located at AA 96 and 283) and two potential N-glycosylation sites (located at AA 133 and 251) present in the AA sequence of the goose CTSD gene, and more importantly, these residues were largely conserved between geese and other species (Fig. 2). Figure 2 also showed that the AA sequence of the goose CTSD gene mainly comprised a signal peptide of 20 AA and two conserved functional domains, including the A1_Propeptide of 37 AA and Cathep-sin_D2 of 325 AA. As shown in Table 2, a homology analysis revealed that both the nucleotide and AA sequences of the goose CTSD gene shared much higher similarities with corresponding homologs from the other avian species compared to those of mammals and fish, particularly with more than 90% similarities at the nucleotide level and more than 96% similarities at the AA level with those of ducks, chickens, and turkeys. These results strongly indicated that the cDNA  Multiple alignments of the deduced amino acid sequences of the CTSD genes from geese and other species. One putative signal peptide and two conserved functional domains (i.e., A1_Propeptide and Cathpsin_D2) were boxed in blue, yellow, and red, respectively. g -indicates active aspartyl residues located at AA 96 and 283. n -indicates the potential N-glycosylation residues located at AA 133 and 251.The GenBank accession numbers of the CTSD genes from other species were shown in Table 2.
isolated from the goose ovary in this study encodes the true goose CTSD gene and is largely conserved with the homologs of other avian species.

Phylogenetic analysis of CTSDs from geese and other species
Based on the AA sequences of CTSDs from geese and another 13 species, a phylogenetic tree was constructed (Fig. 3). The overall topology of this tree suggested that there were obviously three clades among all examined species, including birds, fish, and mammals. In particular, the goose CTSD gene was clustered into the clade of birds and was evolutionarily closest to that of duck CTSD, which was in good agreement with the above results from the homology analysis. Noticeably, in the clade of birds, the zebra finch CTSD gene had a relatively longer evolutionary distance from the others.

Distribution of goose CTSD mRNA in various tissues
The levels of CTSD mRNA were examined in thirteen different tissues of laying geese using real-time PCR. As shown in Figure 4, CTSD mRNA was ubiquitously present in all of the examined tissues but displayed different expression levels. Specifically, the highest levels of CTSD mRNA were detected in the adrenal gland (p<0.05), followed by the liver and heart (p<0.05). Moderate levels of CTSD mRNA were detected in the reproduction-related organs (in-cluding ovarian stroma, oviduct, hypothalamus, and pituitary), spleen, and kidneys, while the lowest levels were detected in the intestine, brain, muscle, and adipose tissue.
Expression profiling of goose CTSD mRNA in the granulosa and theca layers during follicle development Changes in levels of goose CTSD mRNA in the granulosa and theca layers during follicle development were examined using real-time PCR. As shown in Figure 5, goose CTSD mRNA was present in both the granulosa and theca layers of all follicular categories but exhibited different levels between the granulosa and theca layers of the same sized follicles and different expression profiles in either the granulosa or theca layers throughout follicle development. Specifically, CTSD mRNA was much more abundant in the granulosa layer than in the theca layer at all stages of follicle development. Furthermore, the expression of CTSD mRNA was maintained at relatively low and stable levels in the theca layers throughout follicle development (p>0.05). In contrast, as the follicles matured, the mRNA levels of CTSD in the granulosa layers increased gradually from the 2-4 mm to the F5 follicles and reachedmaximum in the F5 follicles (p>0.05), followed by a decline in the F4 follicles and were finally maintained at comparably stable levels in the F4-F1 follicles (p>0.05).  Fig. 3. A phylogenetic tree of CTSDs from geese and other species constructed with the neighbor-joining method using MEGA 7.0. A 1000 bootstrap replication was used to test the reliability of each branch, and bootstrap values were indicated as numbers at the branch nodes. The GenBank accession numbers of the CTSD genes from other species were shown in Table 2.

Discussion
In the present study, the intact CDS of the goose CTSD gene with a length of 1197bp encoding 398 AA was obtained for the first time. Similar to other reported species such as chickens, swine, and rats (BIRCH & LOH 1990;MEI et al. 2008;SHENG et al. 2013), the deduced AA sequence of the goose CTSD gene mainly comprised a signal peptide of 20 AA and two conserved functional domains including the A1_Propeptide of 37 AA and Cathepsin_D2 of 325 AA, which are known as the common features of the lysosomal aspartic proteinases (DUNN 1991), suggesting that goose CTSD functions as a secreted aspartic protease and could have similar physiological actions to the homologs of other vertebrate species. Meanwhile, two potential N-glycosylation sites were identified in the AA sequence of goose CTSD, which further supported that CTSD is a secreted protease because proteins that enter the secretory pathways are generally modified by N-glycans (ANELLI & SITIA 2008). Like other aspartic proteinases, CTSD is a bilobed molecule, and each of the two evolutionary related lobes contributes to one active-site aspartic acid residue (DUNN 1991). It was predicted that the Cathepsin_D2 domain of goose CTSD contained two strongly conserved aspartic acid residues, whose locations were similar to their counterparts in other vertebrate species, suggesting that these residues could be essential for substrate binding and catalysis. Besides, results from homology and phylogenetic analyses suggested that goose CTSD shared relatively higher AA sequence similarities with the homologs from other avian species, especially more than 96% similarities with those of ducks, chickens, and turkeys and was evolutionarily clustered into the avian clade. These results strongly demonstrated that the cDNA isolated from the goose ovary in this study encodes the true goose CTSD gene and suggested functional conservation of avian CTSDs.
To preliminarily explore the physiological actions of the goose CTSD gene, its mRNA expression levels in various tissues were determined.It was observed that goose CTSD mRNA was ubiquitously expressed in all of the examined tissues, indicating that CTSD is a versatile protease that regulates the development and functions of almost all tissues and organs. Specifically, goose CTSD mRNA was much more abundant in the adrenal gland, liver, and heart, which is indicative of a potential role for CTSD in maintaining metabolic processes such as lipid homeostasis (KHURANA et al. 2019). Notably, moderate levels of goose CTSD mRNA were detected in the reproductive organs, including the ovarian stroma, oviduct, hypothalamus, and pituitary, implying its important roles in regulation of female reproductive activities because the hypothalamic-pituitary-gonadal/adrenal (HPG/A) axes are known as the central regulators of the development and function of the reproductive system (WINGFIELD & SAPOLSKY 2003). In view of this, expression of goose CTSD mRNA in the granulosa and theca layers of ovarian follicles at different developmental stages was further determined. The ubiquity of goose CTSD mRNA in both the granulosa and theca layers of all follicular categories potentiated its roles in regulating follicular cell functions. Considering that in oviparous species vitellogenesis is essential for oocyte and embryonic development by providing them with energy and essential nutrients such as amino acids, lipids, carbohydrates, vitamins, phosphorus, and sulfur (ROMANO et al. 2004) and that CTSD is recognized as a lysosomal aspartic protease crucial for protein degradation and lipid metabolism (HAIDAR et al. 2006), CTSD produced by the granulosa and theca cells was proposed to have a role in the degradation and absorption of liver-synthesized yolk precursors during avian follicle development. In support of this, it was evidenced that CTSD is a key enzyme responsible for yolk formation in chicken ovaries (RETZEK et al. 1992). Furthermore, levels of goose CTSD mRNA were much higher in the granulosa layers than in the theca layers throughout follicle development, and moreover, its levels remained almost constant in the theca layers but changed significantly in the granulosa layers as the follicles matured, potentially suggesting a predominant role for CTSD in regulating granulosa cell activities. It is widely accepted that granulosa cell apoptosis is the principal cellular mechanism of avian ovarian follicular atresia and the susceptibility of granulosa cells to apoptosis depends on the stages of follicle development (JOHNSON et al. 1996;JOHNSON 2003). Moreover, CTSD has been demonstrated to be involved in regulating multiple cellular activities including apoptosis (MINAROWSKA et al. 2007). Thus, it was postulated that the varying mRNA levels of the goose CTSD gene in the granulosa layers could be closely related to the fate of granulosa cells and thereby the process of follicle development. Of note, the mRNA levels of the goose CTSD gene in the granulosa layers reached a maximum in the F5 follicles and were significantly higher than in the 8-10 mm follicles, which suggested that CTSD may have a role in follicle selection by regulating granulosa cell activities, a process through which a single follicle among the 8-10 mm cohort is selected to enter the preovulatory hierarchy while the non-selected follicle will cease growth and eventually undergo atresia (JOHNSON & WOODS 2009;JOHNSON 2015;HU et al. 2020).
In conclusion, we have successfully cloned the full-length CDS of the goose CTSD gene (GenBank accession No.: MT812700) and described its structural characteristics, primarily consisting of a signal peptide and two conserved functional domains including the A1_Propeptide and Cathepsin_D2. The high sequence similarities of the CTSD genes in avian species suggested functional conservation of avian CTSDs, and the varying levels of CTSD in the granulosa layers could be closely related to the process of follicle development possibly by regulating both the degradation and absorption of yolk precursors and granulosa cell apoptosis. However, the exact roles of the CTSD gene in avian follicular cells as well as its underlying mechanisms remain to be further elucidated.

Author Contributions
Research