The goal of this study was to identify differentially expressed ovarian genes during the little studied periods of primary and early secondary oocyte growth in coho salmon. A number of differentially expressed genes were successfully identified through SSH and qPCR that are known to play important roles during oogenesis, such as zona pellucida development, sequestration and processing of lipoproteins, cell cycle control, and the fertilization response. Interestingly, some genes involved in vitellogenesis, fertilization, and embryogenesis were more highly expressed during primary growth than early secondary growth. This pattern is intriguing because proteins encoded by many of these genes are not thought to be utilized until 1–3 years later in this species. Through work mainly conducted in Xenopus it has been well documented that maternal RNAs are deposited in the oocyte early in oogenesis and then stored as messenger ribonucleoprotein particles (mRNPs), which contain proteins such as Y-box proteins and DEAD-box RNA helicases that mask RNA from the translational apparatus [5, 27, 28]. In the present study, the ccne transcript was upregulated during primary growth and would likely represent a maternal RNA based on its role in early embryogenesis (see below). However, other transcripts, such as vldlr and cathepsins, associated with later aspects of oogenesis itself were also highly expressed during primary growth. Although protein data are necessary to resolve this issue, one possible explanation for the early transcription of such genes is that some oogenesis related mRNAs are subject to masking during early oogenesis as described for traditional maternal RNAs.
Other transcripts such as lpl, apoe, lcal, rdh1, amh, gsdf, and fshr increased significantly during early secondary growth when CA are abundant in the oocytes, but lipid droplets are not yet evident. Some of these transcripts were likely derived from the follicle cells and showed a dramatic increase across stages (2 to 6 fold). These data together with results of previous studies discussed in detail below, suggest an increased role of the follicle cells during early secondary growth in coho salmon and potential regulation of this transition by FSH and TGFβ family peptides.
Based on GO annotation and examination of the literature, the majority of genes revealed by SSH were of oocyte origin. The SSH method employed enriches for rare transcripts through PCR steps; however it appears that copious mRNA from the oocytes overshadowed differentially expressed genes originating from the follicle/interstitial cells. This idea is supported by the observed differences in abundance of follicle cell transcripts, such as amh and gsdf, which were candidate genes obtained from a follicle/interstitial cell enriched library . As first noted by Goetz and colleagues  these findings suggest that it is necessary to enrich for these cell layers prior to SSH to increase the likelihood of revealing rare transcripts of the granulosa or theca/interstitial cells.
Analyzing transcript levels across stages of oogenesis
One problem with studying the ontogeny of ovarian gene expression, especially in oviparous vertebrates with large eggs, is the dramatic change in oocyte size and RNA composition that occurs during oogenesis. Changes in the size and number of follicles per tissue mass and instability of housekeeping genes complicate across stage comparisons making it difficult to interpret transcript abundance data in a biologically relevant way [e.g., [21, 31–34]]. To gain insight into this problem, we quantified RNA yields per tissue mass and per follicle, and transcript abundance in total RNA and mRNA preparations. As oogenesis progressed, total RNA yielded per tissue mass and the proportion of mRNA relative to non-polyadenylated RNA declined (Table 3). Furthermore, the yield of RNA per follicle was higher in more advanced follicles but the amount of mRNA did not increase proportional to follicle size. This change in RNA composition had a significant effect on apparent transcript levels for a variety of genes expressed in the oocyte and follicle cells depending on whether mRNA or total RNA was used as template for cDNA synthesis. Housekeeping genes, such as ef1a, were similarly expressed across stages when mRNA was used as the template, irrespective of whether data were calculated per unit template RNA or per follicle. Our results indicate that if total RNA is used as a template without normalization to a housekeeping gene one can get misleading results, such as an apparent decline in transcripts that are actually stably expressed, an accentuated decline in downregulated genes, or no change in transcripts for upregulated genes. Results were most skewed in total RNA preparations when presented on a per follicle basis and most closely resembled mRNA based results when normalized to ef1a. In summary, use of mRNA as template and normalization of qPCR data to ef1a generated results that best reflected transcript abundance within the follicle.
Zona pellucida glycoprotein genes
The zona pellucida is the acellular membrane that not only encloses the oocyte, but is also critical to optimal oocyte growth and preventing polyspermy after fertilization. The zona pellucida in vertebrates consists of highly sulfated zona pellucida glycoproteins (ZPs). Four ZP subfamilies, ZPA, ZPX, ZPB and ZPC, have been described and are each found in fish except ZPA [19, 24]. Several coho salmon zp genes were revealed by SSH and all exhibited significantly elevated mRNA levels during primary growth relative to early secondary growth (Fig. 2). Consistent with this, other studies have shown that zp transcripts are abundant in oocytes during early oogenesis and make up a significant portion of the ovarian transcriptome [7–9, 13, 31].
Few studies in fish have focused on the zp-related transcription factor, figla. In mammals, however, figla is required for primordial follicle development and transcription of the zp genes [6, 35]. Like the zp genes, figla is highly expressed in primary oocytes and has been localized to the ooplasm in medaka fish, Oryzias latipes . In the present study, mRNA levels for both the zp genes and figla EST were higher during primary growth. The similar profile of figla and the zp genes in this study along with work in other species [35, 36] suggests that transcription of this family of genes is highly coordinated.
Lipoprotein uptake and processing
Several transcripts associated with vitellogenesis, such as vldlr and vldlro, were highly expressed during primary growth and declined significantly by early secondary growth (Fig. 3). Vldlr has been widely studied and is responsible for uptake of hepatically-derived vitellogenin by the oocyte . On the other hand, very little is known about vldlro, which contains an O-linked sugar domain, is expressed in the ovary and somatic tissues of some fishes [33, 37, 38], and is thought to mediate uptake of lipoproteins other than vitellogenin . Findings of the present study are consistent with work in rainbow trout which showed that transcription of vldlr began shortly after female differentiation  and declined by early vitellogenesis, becoming nearly undetectable by mid-vitellogenesis . Given that vldlr mRNA levels are low during much of the period of active uptake of vitellogenin, it is widely believed that the Vldlr protein is recycled during vitellogenesis [20, 37, 40].
Two genes, lpl and apoe, associated with lipid or lipoprotein uptake by oocytes increased 2–3 fold during early secondary growth. Lpl cleaves fatty acids from plasma lipoproteins and thus facilitates lipid transport across biological membranes, while Apoe is a lipid binding protein that mediates recognition and internalization of plasma lipoproteins by cell surface receptors. The timing of this increase is interesting considering that the CA stage just precedes the appearance of lipid inclusions within the salmon oocyte. Few studies have focused on Lpl and Apoe in the teleost ovary. However, studies in rainbow trout [41, 42] demonstrated that lpl mRNA levels and Lpl activity increased steadily during vitellogenesis and peaked at late vitellogenesis. In sea bass (D. labrax), lpl mRNA was localized to the follicle cells and high lpl mRNA levels and Lpl activity coincided with the appearance of oocyte lipid inclusions . Thus mounting evidence suggests that Lpl may be involved in lipid uptake associated with secondary oocyte growth in fish. To our knowledge, apoe mRNA has been measured in the ovary in only one other study in fish , which demonstrated expression during very early oogenesis. Together these data indicate there is increased expression of genes associated with lipid transport prior to significant lipid incorporation.
Cathepsins are lysosomal enzymes that in the oocyte are responsible for proteolytically cleaving vitellogenin into its constituent yolk proteins and play a role in oocyte reabsorption during atresia [2, 44, 45]. Coho salmon cathepsins revealed by SSH showed different patterns of expression during oocyte growth. The ctsb transcript was more abundant during primary growth relative to early secondary growth. Both ctsd and ctsz mRNA levels were not different across stages and would be considered false positives of SSH, which are commonly encountered with this technique . Ctsd is considered the major protease responsible for cleaving vitellogenin into yolk proteins . However, in the barfin flounder, Ctsb was implicated in this role . In the teleost, Fundulus heteroclitus, ctsz expression was relatively stable during oocyte growth and maturation , and in rainbow trout ctsz was upregulated during maturation  and correlated with egg quality . Data generally suggest that ovarian cathepsins may be regulated post-transcriptionally and thus transcript abundance may not correlate well with cathepsin enzymatic activity [42, 44, 50].
Cortical alveoli components
Studies in Xenopus have shown that 70% of the proteins contained in cortical granules are lectins . In fish oocytes, lectins have a number of biological functions including block to polyspermy at fertilization and defense against pathogens . In the present study, lcal, a Ca2+-dependent or C-type lectin, was more highly expressed at the CA stage and was the predominant transcript from the CA stage SSH library, which is not surprising given the abundance of CA evident in the ovarian histology. Indeed, C-type lectins are often highly represented in ovary transcriptomes in fish [9, 16, 52]. In contrast, however, two other CA components, lrham and alv, were not differentially expressed across stages. Lrham is an oocyte lectin implicated in block of polyspermy that was localized to CA of rainbow trout . Alv is a metalloproteinase first identified in medaka that is released by CA after fertilization to induce zona pellucida (chorion) hardening . Interestingly, when examined as a group, the CA component genes identified in the present study were not transcribed coordinately during oocyte growth as shown in rainbow trout .
Other ovarian transcripts
A subunit of ferritin heavy polypeptide, fth3, was the predominant transcript in the P stage library and qPCR verified that mRNA levels were elevated during primary growth. Ferritins are important because they store and transport iron atoms . Since iron is a critical constituent of metalloproteins, such as enzymes and oxygen carriers, it is essential to all organisms. Free iron, however, can be highly toxic to cells and thus ferritin protects cells from the damaging effects of iron, but makes it readily available. Studies have documented high levels of ferritin subunit mRNAs in the ovary [13, 49] but little is known about their specific role.
Like ferritin, dnaja2 and ccne showed significantly higher transcript levels during primary growth. Dnaja2 encodes a chaperone protein associated with unfolded protein and heat shock protein binding, while cyclins are positive cell cycle regulators that appear to be profuse in the fish ovary [30, 57]. Ccne is transcribed and stored during oocyte growth in goldfish and is thought to be important to the first embryonic cell cycles . The high expression of ccne during primary growth in this study is consistent with its early transcription in goldfish and suggests it is a classic maternal mRNA, as shown for some other cyclins .
Rdh1 and cope showed an opposite profile with transcript levels higher during early secondary growth. Retinol dehydrogenases are involved in the synthesis of retinoic acid, the active form of vitamin A, which regulates cellular growth and differentiation, embryogenesis, and reproduction in vertebrates . Because of the diverse functions of retinol dehydrogenases, it is unclear what role Rdh1 may play during previtellogenic growth. However, based on the recent detection of other retinoid-related transcripts, such as retinol dehydrogenase type II and retinol binding protein in the trout ovary , several players in this cascade are present during oogenesis and likely play an important role during oogenesis and/or early embryogenesis.
The cope transcript encodes the epsilon subunit of a coatomer complex protein. One well characterized coatomer protein is clathrin, which is involved in receptor-mediated endocytosis and the transport of proteins from the Golgi network . At this point it is not possible to determine what process the coatomer complex gene identified here is associated with, but perhaps it is involved in uptake of vitellogenin by receptor-mediated endocytosis, protein trafficking, and/or the immune response.
Transcripts for fshr, amh, gsdf, and lpl, which are all likely produced in follicle cells, exhibited the largest change in abundance across the P and CA stages with levels increasing 4–5 fold. The increase in fshr during this transition from primary to secondary growth together with previous data showing a progressive increase in plasma FSH from the P to lipid droplet stage and effects of FSH on ovarian steroidogenesis [21, 61–63], suggest FSH plays an important role in the endocrine control of this phase of oogenesis in coho salmon. It is not known, however, whether the expression of follicle cell transcripts such as those identified in this study is regulated by FSH and/or other endocrine or paracrine factors.
Transcript levels for two members of the TGFβ superfamily, amh and gsdf, also increased 4–5 fold from the P to CA stage. In female mammals, AMH is produced in the granulosa cells, increases at puberty onset reaching peak levels in small antral follicles, diminishes in later stages, and is no longer detectable during the FSH-dependent final stages of follicle growth or in atretic follicles . Recently, the structure of Amh has been characterized in fish [65, 66] and studies have primarily focused on its expression during sex differentiation [39, 65]. During oogenesis in zebrafish, amh has been localized to granulosa cells where transcript levels peak at the CA stage and progressively decline at onset of yolk incorporation, reaching non-detectable levels by late vitellogenesis. Gsdf is a recently identified gonad-specific cytokine that appears to exist only in teleosts . In rainbow trout, gsdf mRNA was localized to somatic cells of the genital ridge during embryogenesis and Sertoli and granulosa cells during gametogenesis . Gsdf plays a role in primordial germ cell and spermatogonial proliferation, but its role in the ovary is unclear, as it did not induce oocyte proliferation in trout. Based on increased gsdf transcript levels during secondary growth in coho salmon and the ability of Gsdf to stimulate germ cell proliferation in trout, it is possible this factor plays a role in granulosa cell proliferation that occurs during this period.