Digital image analysis of lipid and protein histochemical markers for measuring oocyte development and quality in pearl oyster Pinctada mazatlanica (Hanley, 1856).

ABSTRACT We applied quantitative histochemical techniques and digital image analysis to study seasonal cycles of use of lipid and protein reserves during vitellogenesis in the pearl oyster Pinctada mazatlanica. Female gonad samples were collected seasonally during an annual cycle and processed histologically to characterize the gametogenic cycle, analyze variations in the frequency and size of vitellogenic and postvitellogenic oocytes and calculate the ooplasm:nucleoplasm ratio for both types of oocytes. Lipid and protein inclusions in each type of oocyte were identified using Sudan Black B and Schiff's ninhydrin stains. In both cases, quantification of lipid and protein components was performed through measuring variations in the color coverage area of the oocyte with a digital image analysis system. With this procedure, we calculated a lipid index and a protein index to refer oocyte quality. The lipid index was higher in winter, suggesting a strategy towards storage in the gonad. The protein index was highest during spring in vitellogenic oocytes and during winter in postvitellogenic oocytes, indicating that proteins are actively used during oocyte growth. These results, together with data of the ooplasm:nucleoplasm ratio, suggest differential accumulation of lipid and protein components within the ooplasm during oocyte development. Quantitative histochemistry and digital image analysis represent a combination of reliable techniques for evaluating reproductive processes and oocyte growth and quality.

KEY WORDS: digital image analysis, histochemistry, oocyte quality, Pinctada mazatlanica, vitellogenesis, pearl oyster

INTRODUCTION

Since the 1970s, many studies dealing with reproductive physiology of marine bivalves having commercial value, such as Pectinidae, Ostreidae, Mytilidae and Pteriidae have been published (see review by Navarro 2004). Most studies have described cytological characteristics of the gonad, analyzed cycles of use of energy reserves during gametogenesis and evaluated metabolic strategies used by the animals to ensure gamete maturation and quality. These issues are particularly important with female gametes, where high inputs of energy reserves, especially lipids and proteins, are mobilized to the gonad and incorporated into yolk components as the main sustaining means of embryos and larvae (Gallager & Mann 1986, Gallager et al. 1986). Therefore, lipids and proteins, especially the former, have been used as indicators of oocyte quality and/or broodstock condition (Fraser 1989, Barber & Blake 1991, Racotta et al. 1998, Racotta et al. 2003, Rodriguez-Jaramillo 2004).

Traditionally, lipids and proteins in tissues of diverse marine animals are quantified using analytical biochemical methods (e.g., Bligh & Dyer 1959, Bradford 1976). Unfortunately, these techniques may lead to misinterpretation of data from gonadal tissue because the results show information of the amount of lipids and proteins present in all parts of the tissue, including interstitial connective tissue matrix, mucopolysaccharide and collagen-fibers layer and, if present, other kind of diverse storage cells, such as vesicular connective tissue (VCT) cells, adipogranular (ADG) cells, auxiliary cells, etc. Similarly, conventional histochemical techniques can only qualify the presence (referred to as "+") or absence (referred to as "-") of protein and lipid components in the gonad, but cannot measure their concentration. In contrast, histochemical techniques, when combined with digital image analyses (DIA), can accurately discriminate between the different tissues and cells forming the gonad. This makes quantification of the content of lipid and protein reserves within individual oocytes possible and advances the value of oocyte studies. Because of their recent development, quantitative histochemical techniques (QHT) and DIA have been applied in only a few studies, basically to measure the area of visceral mass occupied by gonadal tissue (Heffernan & Walker 1989, Delgado & Perez-Camacho 2003), and more recently, to calculate indices that measure changes of lipid content of oocytes during oogenesis (Rodriguez-Jaramillo 2004).

When dealing with overexploited, high-commercial value species, such as pearl oysters, QHT and DIA are helpful methods in reproduction investigations, because information relative to oocyte growth and quality is needed to determine the nutritional requirements of broodstock matured at hatcheries for producing spat. To generate data that will allow a broader understanding of the reproductive physiology of the pearl oyster Pinctada mazatlanica (Hanley 1856), this study used QHT and DIA to evaluate seasonal changes of lipid and protein composition of oocytes during vitellogenesis. This information will help define useful indicators of oocyte quality and more reliable methods for large-scale hatchery culture of this species.

MATERIALS AND METHODS

Gonad Collection

Fifteen organisms ranging 130-140 mm shell height were collected In April, August and December 2002 and April 2003 to cover an annual cycle. The collection site, a submarine shelf (trestle), is located in Bahia de La Paz (24[degrees]16'N, l10[degrees]19'W:. Water temperature was recorded at the time of collection at the site. The number of samplings was limited because P. mazatlanica is classified by Mexican Legislation as a species "Under Special Protection" and collecting permits are restrictive. In addition, the reproductive cycle of this species has been previously studied histologically for this area and the nature of the main reproductive events is already well known; our intent was only to confirm the timing of such events. Collected oysters were taken to the laboratory, scrubbed to remove encrusted epibiota and dissected to excise gonadal tissue. Only females were used in this study.

Histological and Histochemical Analysis of Gonads and Oocytes

Part of the female samples was preserved in a 1:1 (v/v) formalin-calcium chloride mixture for lipid preservation and measurement (Bayliss 1984). The other part of samples was fixed in Davidson solution for protein analysis. Preserved samples were then dehydrated, embedded in paraplast and thin-sectioned (3 [micro]m) along an anteroposterior plane (Howard & Smith 1983). Thereafter, Sudan Black B (SBB) and Schiff's ninhydrin (NIN) histochemical techniques were used to identify lipid and protein elements, respectively (Spannhof 1966). Finished slides were examined with a compound microscope. The resulting images were stored in a computer using a digital image system (Media Cybernetics) equipped with Image Pro Plus (v. 4.5).

As a first approach, gonad samples were classified as either inactive, actively developing, ripe, partially spawned and/or spent, following the conventional scheme defined for pearl oysters by Saucedo & Monteforte (1997) and Saucedo et al. (2002a, 2002b).

Additionally, oocytes were classified according to their vitellogenic stage as previtellogenic (small-immature oocytes lacking yolk), vitellogenic (still immature but growing peduncle-shaped oocytes) and postvitellogenic (fully ripe, free polygonal-shaped oocytes) following the criterion proposed by De Gaulejac et al. (1995). Although all types of oocytes were counted, only vitellogenic and postvitellogenic oocytes were used to evaluate the process of yolk formation.

Seasonal variations in the frequency occurrence and size of both types of oocytes were evaluated in three randomly selected areas of the ovary (Grant & Tyler 1983). With regard to size, 30 oocytes of each type were counted per ovary area and measured manually, drawing the contour of the cell with the aid of the computer mouse. The mean area ([micro][m.sup.2]) of the cytoplasm (ooplasm, O) and nucleus (nucleoplasm, N) was automatically calculated by the software. With these data, the O:N ratio was determined for each type of oocyte. Because oocytes change shape as they grow (e.g., round, peduncular and polygonal), the formula of theoretical diameter of Saout et al. (1999) was applied to standardize all the cell-shape data:

TD = [square root of [4A][pi]],

where TD is the theoretical diameter and A is the area of the ooplasm.

Digital Image Analysis

We followed the methodology standardized by Rodriguez-Jaramillo (2004) for the pen shell Atrina maura. Briefly, 30 oocytes per type per sampling were segmented after their histological examination. Segmentation involves measuring and marking the colored area in the oocyte according to the particular stain used (SBB and NIN) to identify the range of hues in which the largest number of pixels is excited. For SBB and NIN, respectively, colors varied from dark blue to black and light pink to fuchsia. The number of pixels helps infer the area ([micro][m.sup.2]) of individual lipid droplets and protein granules present within the ooplasm, as well as the total coverage area of these inclusions in each type of oocyte (Fig. 1a, b). With these data, we calculated two indices of oocyte quality:

Lipid Index (LI) or Protein Index (PI) = (Area covered by component/ooplasm area) x 100

[FIGURE 1 OMITTED]

Statistic Treatment of Data

One-way ANOVA was applied to detect significant differences in theoretical diameter of vitellogenic and postvitellogenic oocytes over time. In addition, correlation analyses (Pearson r) were used to establish the relation between some oocyte features (O:N ratio) and oocyte indices (LI and PI). Previous to these analyses, data were transformed to an arcsine scale. The significance level of all tests was initially set at P = 0.05 (Sokal & Rohlf 1981).

RESULTS

Reproductive Cycle

Female/male sex ratio was 0.42:1 throughout the study. This represents 4-5 females per sampling (avg. 30%). We recorded warm water temperatures during the second half of 2002 and first trimester of 2003 as a consequence of an "El Nino" event. The lowest and highest water temperatures occurred in April (22.5[degrees]C) and August 2002 (29.5[degrees]C) (Fig. 2). The annual reproductive cycle is also depicted in Figure 2. High frequency of ripe ovaries was detected in April (50% at 23[degrees]C) and August 2002 (77% at 29[degrees]C). Although some few specimens spawned in December 2002 (7.5% at 24[degrees]C)--surely as a continuation of a previous spawning occurred from September to November--the massive spawning in this study was recorded in April 2003 during the spring reproductive peak (78.6% at 24[degrees]C). Spent specimens were mostly observed in December 2002 (77%).

[FIGURE 2 OMITTED]

Variations of Oocyte Frequency and Size

Figure 3 shows seasonal variations in the frequency of the different types of oocytes. Vitellogenic and postvitellogenic oocytes were observed throughout the annual cycle, with the former showing a peak in August 2002 (ripe stage) and the latter in April (developing stage) and December 2002 (spent stage). A large number of atresic oocytes (25.1%) were observed only in December 2002.

[FIGURE 3 OMITTED]

Seasonal variations in theoretical diameter of vitellogenic and postvitellogenic oocytes are presented in Figure 4. Larger oocytes of both types were recorded in April 2002 (for lipid and protein analyses). Smaller vitellogenic oocytes were observed in April 2003 (both analyses) and smaller postvitellogenic oocytes were found in December 2002 (proteins) and April 2003 (lipids). There were significant differences in theoretical diameter of vitellogenic (F = 18.3, P < 0.05) and postvitellogenic oocytes (F = 18.5, P < 0.05) over time. Vitellogenic oocytes were significantly smaller and larger in April 2002 and April 2003, respectively; whereas postvitellogenic oocytes were significantly smaller in April and December 2002 and larger in April 2003.

[FIGURE 4 OMITTED]

The values of the O:N ratio of vitellogenic and postvitellogenic oocytes are shown in Table 1. For vitellogenic oocytes, maximum values were observed in April (protein analysis) and December 2002 (lipid analysis). Minimum values for both components were recorded in April 2003. For postvitellogenic oocytes, higher ratios were found in December 2002 for both components and minimum ratios in April (proteins) and August 2002 (lipids). There was a significant correlation between the O:N ratio and both indices in vitellogenic oocytes (r = 0.85, P < 0.05 for LI; and r = 0.86, P < 0.05 for PI). Similarly, in postvitellogenic oocytes the correlation was significant between the O: N ratio and the LI (r = 0.70; P < 0.05) and PI (r = 0.55; P < 0.05).

Variations of Lipid Index, Protein Index and Oocyte Features

Maximum lipid index (LI) values of vitellogenic oocytes occurred in samples collected in December 2002, whereas maximum protein index (PI) values occurred in samples collected in April 2003 (Fig. 5). For both types of oocytes, minimum LI values were observed in April 2002 and minimum PI values in August 2002. There was a significant correlation between both indices and the theoretical diameter of vitellogenic oocytes (r = 0.77, P < 0.05 for LI and r = 0.87, P < 0.05 for PI). For postvitellogenic oocytes, there was also a significant correlation between PI and oocyte diameter (r = -0.23, P < 0.05), whereas the correlation was not significant for LI (r = 0.03, P > 0.05).

[FIGURE 5 OMITTED]

Table 1 shows seasonal variations in mean area and mean diameter of lipid droplets and protein granules present within the ooplasm of both types of oocytes. For vitellogenic oocytes, the highest Ma was observed in April 2002 (proteins) and in December 2002 (lipids), whereas the highest Md was found in April 2002 (both components). For postvitellogenic oocytes, higher Ma values were recorded in April 2002 (both components), and higher Md values occurred in August 2002 (lipids) and April 2003 (proteins). For both types of oocytes, smaller Ma and Md features were seen in August 2002 for proteins and in April 2003 for lipids.

DISCUSSION

In this study, the main reproductive events (e.g., gonad ripeness and spawning, as well as peaks of oocyte frequency and size) were similar to those previously reported for P. mazatlanica in the area of Bahia de La Paz (e.g., Saucedo & Monteforte 1997, Saucedo et al. 2002a, 2002b, Garcia-Cuellar et al. 2004, Vite-Garcia 2005). However, because the second half of 2002 and the first quarter of 2003 were "El Nino" events, there were some differences in the timing of reproductive events. Apart from the well-documented summer reproductive peak, occurring in August in this study when water temperature was 29.5[degrees]C, we also observed unusual high numbers of ripe gonads and postvitellogenic oocytes during the winter season, associated with warm sea temperatures (23[degrees]C to 24[degrees]C). We also found during the winter a high percentage of oocytes undergoing atresia. We believe that these findings are typical examples of short-term, reproductive cycles that run soon after a partial spawning occurs and prevail until environmental conditions are suitable (Saucedo & Monteforte 1997). Hence, the El Nino event produced an alternate cycle from December 2002 through April 2003. We also suggest, in agreement with Dorange & Le Pennec (1989), that atresia appearing during alternate cycles reflect a strategy for recycling nutrients from unspawned residual oocytes to new gametes that rapidly form after a partial spawning. Based on these results, the pearl oyster P. mazatlanica may be considered a conservative species in relation to the use of nutrients during gametogenesis (Bayne 1976).

Our main objective in this study is to define reliable indicators of oocyte quality during growth. Apart from the conventional methods that we used for this purpose (e.g., variations in the frequency and size of oocytes), we used the O:N ratio based on the concept of allometric growth of the ooplasm with regard to the nucleus. Thus, whereas the nucleus stops growing very soon after vitellogenesis commences, the ooplasm continues to expand from the gradual accumulation of nutrients during the vitellogenic phase. Again, we detected the highest values of the O:N ratio during the winter season, coinciding respectively with maximum lipid levels in vitellogenic oocytes and maximum protein contents in postvitellogenic oocytes. These findings, not only suggest differential accumulation of both components into the ooplasm while oocytes are growing, but it indicates that protein and lipid reserves flow through different metabolic pathways related to the energy requirements of the species. Data from previous studies support this possibility. Gabbott (1975), for example, suggested lipogenesis in Mytilus edulis from ingested or stored carbohydrates as a strategy to provide rich energy substrates for the buildup of gametes. Other authors (Gabbott 1975, Desai et al. 1979, Epp et al. 1988, Barber & Blake 1991) state that proteins are basically used during the last phases of gametogenesis before spawning occurs, especially when lipid and glycogen reserves have been depleted. Similarly, Saucedo et al. (2002a) reported an active mobilization of proteins from the muscle to the gonad exclusively from January to June (spring reproductive peak), whereas carbohydrates were only used from July through October (summer peak). Unfortunately, very few authors have used the O:N ratio as a predictive tool of oogenesis progress in marine bivalves (e.g., Rodriguez-Jaramillo 2004), and so far, no clear pattern has been established. Although this ratio seems to be a useful indicator of oocyte development in P. mazatlanica, more research within this context is needed to confirm our findings, especially if the O:N ratio is applicable only when comparing oocytes of the same developmental stage (Rodriguez-Jaramillo 2004).

Differential accumulation and usage of lipid and protein reserves in developing oocytes was confirmed with QHT and DIA. Like biochemical analytical methods, QHT and DIA allowed us quantifying temporal changes in lipid and protein composition of gonadal tissue using the results of the analysis of LI and PI. However, QHT and DIA supported a more accurate method of studying individual oocytes, basically to track and measure variations of the size and number of lipid droplets and protein granules associated with the formation of the yolk molecule within the ooplasm (see Fig. 1a, b). For example, LI values were higher in winter 2002 and dropped in spring 2003 when the main spawning ended and the spent stage started. This pattern supports the idea that lipids, in the form of triglycerides, as identified by Holland (1978) and Gallager et al. (1986), are required in winter as a strategy to recycle nutrients and save energy in storage tissues (e.g., digestive gland) for sustaining future reproductive events, such as the short-term cycles mentioned earlier. This scenario also agrees with observations by Desai et al. (1979) for P. fucata, Besnard (1991) for Pecten maximus, Barber & Blake (1991) for Argopecten irradians and Saucedo et al. (2002a) for P. mazatlanica that lipid levels in the gonad drop after spawning and raise again when organisms are nearly ripe. In contrast, PI values were lower than LI values, outlining 2 important aspects of the metabolic regulation of the species throughout oogenesis: first, that the protein fraction of the yolk molecule in the oocyte is less abundant than the lipid fraction (Fig. 1 a, b), as opposed to traditional biochemical data showing that protein, because the most conspicuous element of soft tissues in marine bivalves is always present in higher concentrations than total lipids (see Besnard 1991, Racotta et al. 1998, Saucedo et al. 2002a, Racotta et al. 2003). Second, that protein reserves, which may have their origin in the adductor muscle, are stored in other cell or tissue compartments of the gonad separate from the oocyte, such as connective tissue and collagen fiber matrices, auxiliary cells, VCT cells and/or the Balbiany body. Although the presence and role of these compartments has been reported in P. mazatlanica by Saucedo et al. (2002b), their temporal changes during oogenesis have not been measured.

Because the use of QHT and DIA techniques for the study of reproductive biology of marine bivalves is so recent, comparative inter and intraspecies data are limited. So far, QHT and DIA have been used to calculate indices (e.g., gonadal occupation index, lipid index) that helped measure the degree of sexual maturity in Ruditapes decussatus (Delgado & Perez-Camacho 2003) and estimate the lipid content of yolk of developing oocytes through comparing wild versus hatchery conditioned A. maura broodstock (Rodriguez-Jaramillo 2004). With P. mazatlanica, the results of this study are being applied to commercial activities where broodstock conditioning is a crucial factor in achieving oocyte quality, larval vigor and survival and successful production of spat outside the main reproduction season. However, because our knowledge about the metabolic control of reproduction in pearl oysters is still limited, additional studies with QHT and DIA are required to quantify temporal changes in other storage tissues (e.g., digestive gland, mantle) and/or storage cells (e.g., VCT, auxiliary cells) during oogenesis and spermatogenesis. Similarly, these techniques can be applied to measure seasonal variations in other oocyte components (e.g., phospholipids) or to evaluate the process of membrane and cell degeneration during atresia.

ACKNOWLEDGMENTS

The authors thank Horacio Bervera and Juan Jose Ramirez at CIBNOR for diving support during collection of samples and Ira Fogel at CIBNOR for improving the English language of the manuscript, and they are also grateful to Hector Acosta-Salmon (James Cook University, Australia) for valuable comments and suggestions to improve the quality of the manuscript. This work was part of an institutional project of CIBNOR.

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ELIANA GOMEZ-ROBLES, CARMEN RODRIGUEZ-JARAMILLO AND PEDRO E. SAUCEDO * Centro de Investigaciones Biologicas del Noroeste (CIBNOR). Mar Bermejo 195, Col. Playa Palo de Santa Rita, La Paz, B. C. S., 23090, Mexico

* Corresponding author. E-mail: [e-mail]

TABLE 1.
Cytological features of vitellogenic and postvitellogenic
oocytes of Pinctada mazatlanica measured with QHT and
DIA during reproductive cycle 2002-2003.

                          Vitellogenic oocytes

                        Lipids

                          Ma                     Md
          O: N         ([micro]              ([micro]m]
                      [m.sup.2])

Apr 02    0.29   208.1 [+ or -] 87.4    270.3 [+ or -] 104.2
Apr 02    0.28   250.1 [+ or -] 153.9   217.0 [+ or -] 99.76
Dec 02    0.33   291.6 [+ or -] 82.9    228.0 [+ or -] 74.2
Apr 03    0.24   184.4 [+ or -] 75.4    193.7 [+ or -] 63.1

                          Vitellogenic oocytes

                       Proteins

                          Ma                     Md
          O: N         ([micro]              ([micro]m]
                      [m.sup.2])

Apr 02    0.28   250.1 [+ or -] 153.9   217.1 [+ or -] 99.8
Apr 02    0.26    66.0 [+ or -] 41.7    145.0 [+ or -] 75.7
Dec 02    0.25   110.0 [+ or -] 49.3    179.9 [+ or -] 57.7
Apr 03    0.25   107.4 [+ or -] 50.4    165.3 [+ or -] 59.2

                 Postvitellogenic oocytes

                        Lipids

                          Ma                     Md
          O: N         ([micro]              ([micro]m]
                      [m.sup.2])

Apr 02    0.33   291.6 [+ or -] 82.9    228.0 [+ or -] 74.2
Apr 02    0.32   287.9 [+ or -] 75.2    229.9 [+ or -] 43.6
Dec 02    0.36   260.7 [+ or -] 77      183.3 [+ or -] 42.6
Apr 03    0.34   145.0 [+ or -] 107.9   149.5 [+ or -] 82.3

                 Postvitellogenic oocytes

                       Proteins

                          Ma                     Md
          O: N         ([micro]              ([micro]m]
                      [m.sup.2])

Apr 02    0.24   184.4 [+ or -] 57.4     193.8 [+ or -] 63.1
Apr 02    0.28    85.9 [+ or -] 49.9    174.07 [+ or -] 86.5
Dec 02    0.38   119.3 [+ or -] 56.5     198.1 [+ or -] 74.5
Apr 03    0.30  145.57 [+ or -] 60.8     245.6 [+ or -] 73.8

QHT = quantitative histochemical techniques; DIA = digital
image analysis; O: N = Ooplasm: nucleoplasm ratio; Ma = mean
area of lipid droplets and/or protein granules; Md = mean
diameter of lipid droplets and/or protein granules; Values
are mean [+ or -] SE