Changes in Lipid Content of Oocytes of the Penshell Atrina maura as a Criterion of

Gamete Development and Quality: A Study of Histochemistry and Digital Image

Analysis
Author(s) :Marco A. Angel-Dapa, Carmen Rodríguez-Jaramillo, Carlos J. Cáceres-Martínez and Pedro E.
Saucedo
Source: Journal of Shellfish Research, 29(2):407-413. 2010.
Published By: National Shellfisheries Association
DOI: 10.2983/035.029.0217
URL: http://www.bioone.org/doi/full/10.2983/035.029.0217

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Journal of Shellfish Research, Vol. 29, No. 2, 407–413, 2010.

CHANGES IN LIPID CONTENT OF OOCYTES OF THE PENSHELL ATRINA MAURA

AS A CRITERION OF GAMETE DEVELOPMENT AND QUALITY: A STUDY OF
HISTOCHEMISTRY AND DIGITAL IMAGE ANALYSIS

MARCO A. ANGEL-DAPA,1 CARMEN RODRÍGUEZ-JARAMILLO,1

CARLOS J. CÁCERES-MARTÍNEZ2 AND PEDRO E. SAUCEDO1*
1
Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Mar Bermejo 195, Col. Playa Palo de
Santa Rita La Paz, BCS 23096, Mexico; 2Universidad Autónoma de Baja California Sur (UABCS),
Km. 5.5, Carretera al Sur, La Paz, BCS 23000, Mexico

ABSTRACT Tools of histochemistry and digital image analysis were used to quantify changes in the coverage area of lipid
droplets (lipid content) of oocytes of the penshell Atrina maura during oogenesis and to determine its relation to changes in water
temperature and seston content. These data led to calculating a lipid index as a criterion of gamete development and quality.
Gonads were collected monthly for 18 mo and prepared for histochemical processing with Sudan Black B for identification of
lipids. Finished slides were digitized for determining stages of oogenesis and variations in the size of oocytes. Two periods of
greatest reproductive activity occurred during the study, with a lower peak from November through January (;15°C; 26 mg/g)
and a major peak from April through June (;20°C; 25–40 mg/g). Oocyte area significantly varied during the stages of active
development (516–2,743 mm2), ripeness (1,073–2,930 mm2), spawning (145–2,939 mm2), and atresia (331–2,001 mm2). Lipid
incorporation into oocyte cytoplasm followed a clear seasonal pattern, peaking again in winter and spring. Temporal variations in
the lipid index and its relation to oocyte diameter were irregular, but also peaked in winter and spring. Histochemistry and digital
image analysis resulted in reliable methods for estimating oocyte development and quality in this species, and can certainly be
applied in studies of reproduction of other bivalve, invertebrate, and vertebrate species.

KEY WORDS: Atrina maura, digital image analysis, histochemistry, lipid content, lipid index, oocyte quality

INTRODUCTION maura (Rodrı́guez-Jaramillo 2004), and Crassostrea corteziensis

(Rodrı́guez-Jaramillo et al. 2008).
In marine bivalves with a subtropical or temperate distribu-
Regardless of the species, lipid composition of body tissues,
tion, availability of food is limited in time and space, and causes
such as gonads, is determined through conventional analytical
gametogenic cycles to be closely coupled to biochemical cycles
methods (e.g., Bligh & Dyer 1959, Barnes & Blackstock 1973).
to ensure adequate provision of nutrients for reproduction (see
Although these methods are accepted and standardized, they
reviews by Barber and Blake (2006) and Saucedo and Southgate
provide a gross indicator of lipid content (LC) of the entire
(2008)). In these areas, overall development of the gonad depends
gonad tissue, but not of the LC of the different germinal, stor-
on complex interactions of exogenous and endogenous cues
age, and nourishing cell compartments forming the gonad,
prevailing at a certain time and at a certain area, but not always
including male and female gametes, connective tissue, muco-
resulting in viable larvae having sufficient energy reserves to
polysaccharides, and, if present, vesicular connective tissue cells,
complete metamorphosis and settle down (Gallager and Mann
adipogranular cells, and auxiliary cells (Mathieu & Lubet 1993).
1986, Gallager et al. 1986, Le Pennec et al. 1990). Consequently,
Because of the methodological limitation related to tissue
maximizing the quality of developing gametes, especially oo-
compartmentalization, conventional analytical methods may
cytes, is crucial as the best strategy for enhancing vigor and
provide inaccurate information of the real lipid composition
viability of the resulting embryos and larvae, and ensuring
of the gonad, because the other cell compartments may store
survival of the offspring (Dorange & Le Pennec 1989).
energy reserves such as glycogen. In recent years, the incorpora-
Generally, the quality of growing oocytes is measured by the
tion of digital image analysis in histological and histochemical
amount of proteins, lipids, and carbohydrates incorporated into
procedures has allowed studying and quantifying key processes
cytoplasm during yolk formation (Holland 1978). Among these
of gametogenesis, such as the loss of cytoplasm volume during
reserves, lipids are known to play a key role in maximizing the
sperm maturation and the rate of lipid incorporation within
rate of hatched eggs (fecundity), rate of larval growth and survival,
oocytes during vitellogenesis. This newer approach has provided
and rate of settled postlarvae (reproductive success) (Gallager et al.
more reliable criteria for estimating gonad development and
1986, Fraser 1989, Palacios et al. 2007). Because of this, lipids have
quality in some marine bivalve species, which in turn is helping
been considered a reliable indicator of oocyte quality in species
to optimize the protocols for the rearing of spat at the hatchery.
such as Mercenaria mercenaria (Gallager et al. 1986), Crassostrea
This is true for A. maura broodstock matured at different
virginica (Heffernan & Walker 1989), and Ruditapes decussatus
temperatures (Rodrı́guez-Jaramillo 2004) and wild Pinctada
(Delgado & Pérez-Camacho 2003). Similarly, lipids have been
mazatlanica and C. corteziensis during gametogenesis (Gómez-
reported to be very useful for estimating the condition of larvae of
Robles et al. 2005, Rodrı́guez-Jaramillo et al. 2008; Gómez-
species that include Pecten maximus (Le Pennec et al. 1990), Atrina
Robles & Saucedo 2009). For other bivalves and conditions,
however, the value of these criteria has not yet been determined.
We used tools of histochemistry with digital image analysis
*Corresponding author. E-mail: psaucedo04@cibnor.mx to measure changes in LC of oocytes of the penshell A. maura

407
408 ANGEL-DAPA ET AL.
(Sowerby, 1835) during oogenesis. The data helped prepare
a lipid index (LI) to test the hypothesis that this cue provided
a reliable criterion of oocyte (gamete) development and quality.
The study is useful because the genus Atrina is receiving growing
acceptance in national and international markets because of the
large size and appealing flavor of the adductor muscle (Cardoza-
Velazco & Maeda-Martı́nez 1997).
MATERIALS AND METHODS
Origin of Gonads and Data for Field Analysis and Condition Indices
From March 2000 through October 2001, 15 penshells (171 ±
31 mm shell height) were collected each month from Laguna
San Ignacio, Mexico (26°43#N, 113°08#W). Water temperature
was continuously recorded at the collection site with an analog
thermometer (±0.1°C). At each monthly collection, triplicate
water samples were collected to measure availability of food,
as standardized by Luna-González et al. (2000). Briefly, water
was filtered through Whatman glass microfiber filters (model
Z242330; Sigma, St. Louis, MO), rinsed with ammonium
formate, dried at 100°C, burned at 450°C, and weighed
(±0.001 g). The results were used for estimating total seston
and, by extrapolation, the fractions of organic and inorganic
seston.
During each sampling, the specimens were cleaned of
epibiota, shell height and length were measured (±0.1 mm),
and wet weight with shell was recorded (±0.1 g). After mea-
surement, the entire visceral mass (meat) and adductor muscle
were excised from each specimen, and wet weight (±0.1 g) was
recorded. These data were later used to determine a condition
index (CI) and muscle index (MI) as described by Barrios-Ruı́z
(2005).
CI ¼ ðWM=WSÞ 3 100
where WM is the weight of the visceral mass or meat and WS is
the weight of the animal with shell, and
MI ¼ ðWA=WSÞ 3 100
where WA is the weight of the adductor muscle and WS is the
weight of the animal with shell.
Samples of female gonads were also excised and their wet
weights were recorded (±0.1 g). These samples were fixed in
a calcium–formaldehyde (1:1 ratio) solution for 48 h for proper
preservation of lipids (Bayliss 1984).
Histochemical Analysis of Gonads and Oocytes
Preserved samples of female gonads were dehydrated, placed
in embedding media (Paraplast X-TRA; McCormick Scientific,
St. Louis, MO), and ‘‘thin sectioned’’ to 3 mm (Howard & Smith
1983). Slide specimens were stained with Sudan Black B to
identify neutral lipids (triglycerides) (Bayliss 1984). Finished
slides were observed under a standard light microscope (20 3 ,
40 3 ). The gonads were classified by developmental stage as
inactive, developing, ripe, partially spawned, and spent, as de-
scribed by Enrı́quez-Dı́az et al. (2003) and Barrios-Ruı́z (2005).
Oocytes were classified according to their vitellogenic stage
as either previtellogenic (small, immature, lacking yolk), vitel-
logenic (still immature but growing, peduncle shape), and
postvitellogenic (fully ripe, free polygonal shape), following
the criterion of De Gaulejac et al. (1995) for the penshell Pinna
nobilis. Only vitellogenic and postvitellogenic oocytes were
considered, and only when the nucleus was complete.
Lipid Content and Lipid Index of Oocytes
The techniques used for determining LC and LI of oocytes
were developed and standardized at Centro de Investigaciones
Biológicas del Noroeste by Rodrı́guez-Jaramillo (2004). The
procedure consists of randomly selecting 3 areas of the ovary
and digitizing images of 30 oocytes per area. As the first step,
vitellogenic and postvitellogenic oocytes were grouped by size
range, as proposed by Snedecor and Cochran (1989). The
images were archived and processed with Image Pro Plus
(version 5.1; Media Cybernetics, Bethesda, MD) to quantify
the number of lipid droplets occupying the oocyte cytoplasm,
referred to as the ‘‘lipid coverage area’’ or LC. An LI was then
determined with these data using the formula of Rodrı́guez-
Jaramillo (2004):
LI ¼ ðAL=OAÞ 3 100
where AL is the area covered by lipid droplets within the oocyte
cytoplasm (in pixels) and OA is the oocyte area (in mm2).
Statistical Analysis
Data of oocyte diameter, lipid coverage area (LC), and LI
were initially evaluated for homogeneity of their variances using
the Levene test and one-way analysis of variance for deter-
mining whether significant differences are a function of time
(Snedecor & Cochran 1989). If needed, an a posteriori analysis
with the Tukey test was applied to identify homogeneous or
heterogeneous groups of means. Finally, correlation analyses
between some biological parameters (oocyte size, LC, LI) were
conducted on arc sine transformed data. The significance level
of all tests was set at P < 0.05.
RESULTS
Oogenesis and Its Relation to Environmental Conditions
Changes in temperature and seston concentration followed
a typical seasonal pattern and yielded a direct relationship with
oogenesis (Fig. 1). Gonads undergoing active development
occurred almost all year. The peak of reproductive activity
occurred in spring, when temperature increased to 17°C and
seston levels peaked at 40 mg/g. A lesser peak occurred in late
winter, when temperature was at the annual low (;15°C) and
seston was relatively high (;29 mg/g). Ripe gonads (20–80%
postvitellogenic oocytes) occurred from June 2000 through June
2001, with peaks in November 2000 and April 2001 when
temperature was 15–19°C and seston was 13–40 mg/g. Two
spawning events with 5–30% residual oocytes occurred from
March through October 2000 (16–26°C, 20–30 mg/g) and from
April through July 2001 (17–25°C, 25–30 mg/g). The spent stage
prevailed from June through October 2000 and in October 2001,
with ;11% atresic oocytes.
Oocyte Size and Its Relation to the Condition and Muscle Indices
During oogenesis, oocyte area changed significantly in time
(P < 0.05), following a typical seasonal pattern (Fig. 2). Large
 LIPIDS IN ATRINA MAURA OOCYTES
Figure 1. Developmental stages of gonads in the penshell Atrina maura
in Laguna San Ignacio, Baja California Sur, Mexico, from March 2000
through October 2001, and their relation to environmental conditions.
DEV, active development; RIP, ripeness; SES, total seston, including
particulate organic matter (POM) and particulate inorganic matter
(PIM); SPT, spent; SPW, partial spawning; TEM, water temperature.
, DEV; , RIP; , SPT; , SPW; , TEM; , SES.
oocytes occurred in June 2000 (;1,750 mm2) and April through
June 2001 (;1,900 mm2). Small oocytes occurred in March 2000
(;1,200 mm2) and October 2000 and 2001 (;1,265 mm2).
Growth of oocyte was directly related to changes in the CI
and inversely related to changes in the MI (Fig. 2). Large areas
of oocytes coincided with a high CI and low MI, and small areas
coincided with a low CI and high MI.
The oocyte area also varied markedly during different stages
of oogenesis (Fig. 3): active development, mostly vitellogenic
oocytes (516–2,743 mm2); ripeness, comprising postvitellogenic
oocytes (1,073–2,930 mm2); partial spawning with residual oo-
cytes (145–2,939 mm2); and spent stage, mostly atresic oocytes
(331–2,001 mm2).
Figure 2. Changes in oocyte area (OA) during oogenesis of the penshell
Atrina maura collected in Laguna San Ignacio, Baja California Sur,
Mexico, from March 2000 through October 2001, and its relationship to
the condition index (CI) and muscle index (MI). Values represent the mean
% SD. Means sharing the same superscripts are not significantly different
(P > 0.05).
409
Lipid Content Versus Lipid Index
The LC of oocytes during oogenesis followed a different
trend when analyzed as a function of time (Fig. 4A) and in
relation to size intervals (Fig. 4B). Regardless of their size,
oocytes accumulated more lipid droplets within their cytoplasm
during April through June 2001 (;950), which is consistent with
the period of greatest reproductive activity, maximal seston
concentration (;40 mg/g), moderate water temperature
(;18°C), and highest MI (;15%). This peak did not coincide
with the CI. In contrast, lowest LC values occurred in February
2001 (600) and coincided with the process of gonad resorption
during moments of lowest seston load (;12 mg/g), relatively
low MI (;17%), and high CI (;30%). Regardless of time, the
capacity of oocytes to store lipids was directly related to their
size, that is, larger oocytes stored more lipids and smaller
oocytes stored fewer lipids (Fig. 4B).
Different from clear seasonal trends for the LC, changes in
the LI of oocytes was erratic (Fig. 5A). Despite this, there were 3
peaks in March 2000 (;52), September through November
2000 (;50), and March through April 2001 (;51). These peaks
corresponded to reproductive peaks, when gonads were ripe or
spawned. In contrast, the lowest LI occurred in September
2000, when gonads had regressed or had spawned (;43),
February 2001 (;44), and June (;45).
The relationship between the LI and the area of oocytes was
inverse (Fig. 5B), indicating that a lower LI corresponded to
larger oocytes and a higher LI corresponded to smaller oocytes.
Finally, lipid content of oocytes had a significant linear re-
lationship with the LI (r ¼ 0.62; P < 0.05).
DISCUSSION
Utility of the Approach Using Histochemistry and Digital Imaging
Combining histochemistry and digital image analysis
allowed us to compartmentalize the gonad into its cell compo-
nents to study the dynamics of lipids in the female germinal
component only (oocytes). Of the total lipids, the software
differentiated the fraction of neutral lipids or triglycerides,
discerning how they were allocated in the oocyte cytoplasm
during vitellogenesis. This is useful information, because a large
part of the triglyceride reserves are used to build vitellin/
vitellogeninlike lipoproteins that promote the formation of
the yolk (Arcos et al. 2009) and ensures the success of the
progeny (Gallager & Mann 1986, Dorange & Le Pennec 1989,
Le Pennec et al. 1990). Using tools of digital image analysis with
histochemistry has recently provided (1) finer details of sper-
matogenesis and/or oogenesis of A. maura broodstock matured
at the laboratory with different temperature regimes (Rodrı́guez-
Jaramillo 2004), and of gametogenesis of wild P. mazatlanica
(Gómez-Robles et al. 2005) and C. corteziensis (Rodrı́guez-
Jaramillo et al. 2008); (2) indices of coverage area of male and
female acini as part of the gonad, adenomers as part of the
digestive gland index, and fiber packages as part of adductor
muscle in P. mazatlanica for understanding the cycle of storage
and allocation of protein, glycogen, and lipid reserves between
somatic tissues and the gonad during gametogenesis (Gómez-
Robles & Saucedo 2009); and (3) quantification of the number
of muscle fiber packages and spaces between the tubules of the
digestive gland for establishing the storage capacity of C.
corteziensis juveniles reared at the hatchery under different
410 ANGEL-DAPA ET AL.

Figure 3. (A–D) Changes in range of modal frequency of oocytes during oogenesis of the penshell Atrina maura collected in Laguna San Ignacio, Baja
California Sur, Mexico, from March 2000 through October 2001. AO, atresic oocytes; PO, postvitellogenic oocytes; RO, residual oocytes; VO,
vitellogenic oocytes.

diets and stocking densities, and determination of how the
species responds at a cellular level under normal and stressing
conditions (Mazón-Suástegui et al. 2009).
Influence of the Environment on Reproduction and Condition Indices
Previous to this study, the reproductive cycle of A. maura
was described from 3 sites along the Pacific coast, Acapulco
(Aguilar-Ibarra 1964), Michoacán (Soria-Padilla 1989), and
Oaxaca (Ángel-Pérez 2000)—all tropical locations where water
temperature and food are relatively constant throughout the
year. Conversely, for subtropical areas, such as Laguna San
Ignacio, where this study was conducted, changes in water
temperature and availability of food were marked between
seasons and generated 2 peaks of reproductive activity: a major
peak in April through June 2001 and a lower peak in November
2000 through January 2001. These peaks are very similar to
those reported for the 3 tropical locations (April through July
and November through January), which reflects the plasticity of
the species to adapt to habitats within different climatic zones.
For Laguna San Ignacio in particular, Barrios-Ruı́z (2005)
reported large interannual variations in water temperature and
 LIPIDS IN ATRINA MAURA OOCYTES
Figure 4. (A, B) Variation of lipid content of oocytes of the penshell
Atrina maura as a function of time (A) and increase in size (B). Values
represent the mean % SD.
seston load that makes the seawater oligotrophic in nature and
generates a constant input of organic matter (detritus and
phytoplankton), which can significantly increase gonad and
digestive gland biomass. In contrast, some areas of the Gulf of
California inhabited by the penshell, such as Bahı́a de La Paz,
have clearer waters and less seston, thus generating lower input
of food energy to sustain somatic growth and reproduction
(Luna-González et al. 2000).
The environmental data collected in this study suggest that
oogenesis of A. maura in Laguna San Ignacio occurs primarily
by changes in water temperature and secondarily by changes in
the availability of food, which followed an irregular pattern and
varied largely from 12–40 mg/g. There was, however, very little
relationship between changes in water temperature and seston
concentration throughout the study. According to Barrios-Ruı́z
(2005), the marked seasonal changes in availability of food in
Laguna San Ignacio may be caused by turbulence and resus-
pension of sediments and organic material during tropical
depressions or hurricanes, and long-term oceanographic events
such as El Niño or La Niña.
Seasonal changes in the oocyte area were significant
throughout the year and yielded an inverse relationship to the
MI and CI. In addition, it appears that the combination of high
411
Figure 5. (A, B) Variation of lipid index of oocytes of the penshell Atrina
maura as a function of time (A) and increase in size (B). Values represent
the mean % SD.
MI and low CI from May through June, which occurred when
seston load was high (up to 40 mg/g) and water temperature was
moderate (20–25°C), indicates energy mobilization from the
muscle to the gonad to promote maximum reproductive
activity. This evidence, however, is insufficient to confirm such
a scenario, because other storage tissues, such as the digestive
gland, may be participating, even more actively than adductor
muscle, in the transfer of energy to the gonad during gameto-
genesis. This is true for Pteria sterna from Bahı́a de La Paz
(Vite-Garcı́a & Saucedo 2008, Cáceres-Puig et al. 2009) and
likely for A. maura from Laguna San Ignacio (Barrios-Ruı́z
2005). In fact, only in the Pectinidae is the adductor muscle
recognized as the most important energy repository for re-
production (see reviews by Chantler (2006) and Barber and
Blake (2006)).
Lipid Content Versus Lipid Index
Variations in the LC of oocytes followed a typical seasonal
pattern, with a major peak in April through June 2001, whereas
variations in the LC depicted a constant increasing trend when
analyzed as a function of oocyte size. When related to time, the
412 ANGEL-DAPA ET AL.
result clearly reflects the rate at which lipid incorporates into
oocytes and reaches an optimum, assumed at ;20°C and 25–40
mg/g seston content in April through June. These values suggest
that A. maura uses a conservative strategy to regulate oogenesis
in Laguna San Ignacio. When related to oocyte size, increasing
values of LC suggest that, regardless of time, storage of lipid
reserves occurs gradually and synchronously during oocyte
development until reaching the climax of vitellogenesis. Similar
scenarios were described decades ago as the basis for clarifying
the dynamics of lipids in larvae of many marine invertebrates
(Holland 1978, Fraser 1989), including bivalves (Gallager &
Mann 1986). Our results with oocytes are in agreement with
previous reports by Osada and Mori (2000), who noted that the
fastest rate of oocyte growth in C. gigas occurs during the last
stages of vitellogenesis, which corresponds to the moment of
greater lipid composition within oocytes; and by Arcos et al.
(2009), who noted that the lipid content of C. corteziensis gonads
significantly increases with oocyte size, reflecting a synchronous
relationship with oogenesis.
In contrast to LC, variations in LI were irregular in time
and yielded a clear inverse relationship with oocyte size. Despite
the irregular pattern, the 2 LI peaks are consistent with the 2
reproductive peaks detected histologically. Although the con-
stant decreasing trend in LI was unexpected and appears to
have no simple explanation, we believe it may reflect the way in
which the software deals with the units expressing presence of
lipids, either LC (pixels) or LI (percentage) for comparing the
rate of lipid occupancy within the oocyte versus its total area. In
other words, the proportion of lipid incorporation in the oocyte
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ACKNOWLEDGMENTS
The authors are grateful to Daniela Barrios-Ruı́z for
collecting the specimens, and temperature and seston data
for this study. Ira Fogel (CIBNOR) provided a critical edito-
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Consejo Nacional de Ciencia y Tecnologı́a (CONACYT)
through grant 33482-V.
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