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Comparative Biochemistry and Physiology, Part A 147 (2007) 731 742

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The role of vitellogenin during gestation of Girardinichthys viviparus and


Ameca splendens; two goodeid fish with matrotrophic viviparity
Armando Vega-Lpez a,, Esperanza Ortiz-Ordez b , Esther Ura-Galicia b ,
E. Laura Mendoza-Santana b , Rub Hernndez-Cornejo c , Roxana Atondo-Mexia c ,
Alejandra Garca-Gasca c , Ethel Garca-Latorre d , Maria Lilia Domnguez-Lpez d
a

Laboratorio de Toxicologa Acutica, Departamento de Farmacia, Escuela Nacional de Ciencias Biolgicas, IPN. Carpio y Plan de Ayala s/n,
Col. Plutarco Elas Calles Casco de Santo Toms. D.F. CP 11340, Mxico
b
Laboratorio de Histologa, Departamento de Morfologa, Escuela Nacional de Ciencias Biolgicas, IPN. Carpio y Plan de Ayala s/n,
Col. Plutarco Elas Calles Casco de Santo Toms. D.F. CP 11340, Mxico
c
Laboratorio de Biologa Molecular, CIAD Mazatln, Av. Sbalo Cerritos s/n, Mazatln Sinaloa. CP 82010, Mxico
d
Laboratorio de Inmunoqumica I, Departamento de Inmunologa, Escuela Nacional de Ciencias Biolgicas, IPN. Carpio y Plan de Ayala s/n,
Col. Plutarco Elas Calles Casco de Santo Toms. D.F. CP 11340, Mxico
Received 6 April 2006; received in revised form 23 October 2006; accepted 24 October 2006
Available online 16 November 2006

Abstract
Goodeid fish have matrotrophic viviparity, and unlike lecitotrophic fish, yolk loss forces the female to provide the nutritional requirements for
embryonic development. Vitellogenin (VTG) is the yolk precursor protein synthesized in the maternal liver, but there is only circumstantial
evidence regarding VTG supply during the ontogenesis of bony fish with matrotrophic viviparity. Therefore, the goal of the present study was to
identify and quantify VTG during gestation of the black fin goodeid Girardinichthys viviparus and the butterfly split-fin goodeid Ameca
splendens. Females at different gonadic developmental stages were selected in order to evaluate VTG mRNA expression in the maternal liver
using RT-PCR; VTG quantification in maternal muscle and liver, as well as in the embryos, was done using ELISA, and immunohistochemical
detection of VTG was done in the black fin goodeid. The results suggest that VTG supplies nutrients during embryonic development of both
species, which have different life histories. It is possible that the transition from lecitotrophy to matrotrophic viviparity in bony fish with
intraluminal gestation involved adaptive transition strategies that included changes in the relationship between oocytes and follicular cells, as well
as a gradual loss of VTG synthesis during embryonic development.
2006 Elsevier Inc. All rights reserved.
Keywords: Ameca splendens; Embryonic development; Girardinichthys viviparus; Goodeid fish; Matrotrophic viviparity; Vitellogenin

1. Introduction

This paper is part of the 3rd special issue of CBP dedicated to The Face of
Latin American Comparative Biochemistry and Physiology organized by
Marcelo Hermes-Lima (Brazil) and co-edited by Carlos Navas (Brazil), Rene
Beleboni (Brazil), Rodrigo Stabeli (Brazil), Tania Zenteno-Savn (Mexico) and
the editors of CBP. This issue is dedicated to the memory of two exceptional
men, Peter L. Lutz, one of the pioneers of comparative and integrative
physiology, and Cicero Lima, journalist, science lover and Hermes-Lima's dad.
Corresponding author. Tel.: +52 55 5729 6300x62343; fax: +52 55 53 96 35
03.
E-mail address: avegadv@yahoo.com.mx (A. Vega-Lpez).
1095-6433/$ - see front matter 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.cbpa.2006.10.039

According to Rothchild (2003), viviparity is a reproductive


strategy in which the embryo begins and/or completes its
development at the parent's expense. It is known that goodeid
fish have matrotrophic viviparity (Riehl and Greven, 1993);
which is characterized by internal fertilization and the presence
of trophotaeniae. In Mexico, the black fin goodeid Girardinichthys viviparus and the butterfly split-fin goodeid Ameca
splendens are endemic and endangered fish. These fish species
have a relevant evolutionary importance in the American
Continent, as well as a regional economic importance (Webb,

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A. Vega-Lpez et al. / Comparative Biochemistry and Physiology, Part A 147 (2007) 731742

1998). Daz-Pardo and Ortiz-Jimnez (1985) mentioned a


strong sexual dimorphism and prenuptial courtship causing
morphologic, anatomic, and physiologic adaptations that are
distinct in this group of fish. These adaptations create a clear
evolutionary difference that makes the goodeid fish a model
family of fish with matrotrophic viviparity in which to evaluate
the role of vitellogenin (VTG) during gestation.
In viviparous organisms during oocyte maturation, only
enough yolk is accumulated to sustain the early developmental
states of the zygote (Rothchild, 2003); however, it is important
to remember that vitellogenin is a yolk precursor protein and
supplies most of the nutrients needed during ontogenesis
(Arukwe and Goksyr, 2003). In goodeid fish, a small amount
of yolk is produced (Riehl and Greven, 1993), which is only
able to sustain embryogenesis. Therefore, further nutritional
requirements are provided from the maternal body that reach the
embryo through the trophotaeniae (Turner, 1933). The trophotaeniae is an intestinal elongation or a rectal embryonic process,
which functions as a respiratory and nutrient exchange organ
(Turner, 1940a). The trophotaeniae participates in osmoregulation, excretion, and as a connection between the endocrine and
immunological systems of the mother and embryo (Wourms
et al., 1988). Furthermore, it has intrinsic lytic enzymatic
activity (Lombardi and Wourms, 1985b). Other embryonic
structures, such as the pericardial sac, pharynx, digestive tract,
and finfolds, are presumed to absorb nutrients from the maternal
body (Webb, 1998); nonetheless, this function has not been
demonstrated.
During embryogenesis (intrafollicular gestation), viviparous
fish embryos obtain nutrients from the yolk, while during later
developmental states (intraluminal gestation) the nutrients are
obtained from the epithelium of the ovarian stroma. In different
goodeid species, embryo dry weight increases due to the
presence of exogenous metabolites (mainly lipids and proteins)
have been reported to range from 2700% to 38,700% (Wourms
et al., 1988; Hollenberg and Wourms, 1995). These findings
support a strong matrotrophic viviparity (MacFarlane and
Bowers, 1995). Supplies are used mainly for organogenesis
and as energy sources (Lombardi and Wourms, 1985a). Nevertheless, despite the large amount of current knowledge regarding the reproductive peculiarities of goodeid fish, there is
not enough information concerning the adaptive reproductive
strategies of this fish family.
It has been reported that the transition from lecitotrophy to
matrotrophic viviparity was the result of the loss of vitellogenin
synthesis (Wourms et al., 1988; Rothchild, 2003). Unfortunately, this hypothesis has been taken as fact, even though it has not
been demonstrated.
However, changes in phospholipid levels during gonadic
development of marine fish have been observed (MacFarlane
et al., 1993), and increases in plasma triglyceride, phospholipid,
and calcium concentrations, as indirect indicators of VTG, have
been noted (Tinsley, 1985). Nonetheless, the presence of VTG
during embryonic development in fish with strong matrotrophic
viviparity, such as goodeids, has not been demonstrated.
For these reasons, the goal of the present study was to
identify and quantify VTG during the embryonic development

of the black fin goodeid G. viviparus and the butterfly split-fin


goodeid A. splendens, two endemic, endangered species of
Mexico, and to analyze differences in embryonic development
and VTG synthesis between the two species.
2. Materials and methods
2.1. Fish
Since this study involved a protected species, the collection
of the parent group of fishes was conducted only after the
evaluation and authorization of the project by Mexican authorities (Authorization Num/SGPA/DGVD/02750). Four sampling campaigns were carried out during the year (drywarm
season, rainy, end of rainy, and drycold season). Fish were
collected from locations with low levels of contamination; the
black fin goodeid was collected from reservoirs near the
Texcoco Lake area (State of Mexico), and the butterfly split-fin
goodeid was collected from the spring of El Rincn, Teuchitlan,
Jalisco. The fish were transported to the laboratory alive, and
their weight and length were recorded on arrival to determine
their condition factor (Lagler, 1952). Ten females were selected
from each species and for each developmental stage. Black fin
goodeid developmental stages were determined according to
Daz-Pardo and Ortiz-Jimnez (1985). For the butterfly split-fin
goodeid, developmental stages were determined according to
Ortiz-Ordez et al. (in press) criterion. The females were
sacrificed by quick freezing ( 70 C) followed by cervical
dislocation. Some of the black fin goodeid females in
vitellogenesis and carrying embryos close to birth were selected
and fixed in 4% formaldehyde (W/V). Oocytes and embryos
were separated for gravimetric evaluation (100 2.0 C) of
weight gain during embryonic development. Of note, live
females were maintained under controlled conditions until the
end of gestation.
2.2. VTG quantification
Three females of each species at different ovarian developmental stages were sacrificed as noted above. The third distal
portion of the maternal body (without fins), the liver, the gonad
(including ovisac, stroma, and septum), and the embryos were
harvested separately. Tissues were homogenized (8000 rpm) in
PBS pH 7.5 with protease inhibitor (aprotinin 3 g/mL),
centrifuged at 1500 g (5 min), and the supernatant was stored at
70 C until use. VTG quantification was performed according
to Vega-Lpez et al. (2006). Briefly, a hybrid ELISA was used
with rabbit anti-rainbow trout VTG (Oncorhynchus mykiss)
polyclonal antibodies. Calibration curves for the black fin
goodeid (gvVTG) and the butterfly split-fin goodeid (as VTG)
with 1 to 30 ng of VTG were used. For VTG quantification,
5.0 L of supernatant were added to 100 L of anti-VTG
polyclonal antibody (1:100) according to the protocol; each
sample was run 6 times during 3 consecutive days. Total protein
concentration was determined (Bradford, 1976) using bovine
serum albumin as the standard. A two-way ANOVA with a
TukeyKramer test (p 0.05) was used in order to analyze the

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VTG concentration data. The details of VTG purification, VTG


characterization, AgAb cross-reactions, and the hybrid ELISA
quality control aspects that were used have been previously
reported by Vega-Lpez et al. (2006).
2.3. Immunohistochemical detection of VTG
This analysis was performed only in the black fin goodeid
females at different gonadic developmental stages given that
previous VTG quantification of the butterfly split-fin goodeid
has been found to be one order of magnitude below that of the
black fin goodeid; thus, the antigenantibody reaction would
have been difficult to detect. The intact gonads were fixed in 4%
formaldehyde (W/V) in PBS pH 7.5 for 1 week and then
paraffin-embedded. Serial sections of 8 m were obtained.
Next, immunohistochemical detection of VTG according to a
modification of the method proposed by Rasmussen et al.
(2002) was done. First, paraffin was removed using xylene
followed by absolute ethanol. Endogenous peroxidase activity
was eliminated with 10% hydrogen peroxide in absolute
methanol for 30 min. The sections were then re-hydrated with
decreasing concentrations of ethanol to PBS-T (PBS + 0.05%
Tween-20). Sections were blocked for 60 min with 0.25%
gelatin in PBS (blocking solution), and then incubated at room
temperature for 60 min with anti-VTG polyclonal antibody
(1:100) in blocking solution. After washing, the sections were
incubated for 60 min with goat anti-rabbit IgG (1:1000, Sigma)
coupled to horseradish peroxidase (HRP) in blocking solution
with a further wash. VTG was visualized after incubating for
5 min with the substrate (0.04% 33 diaminobenzidenitetrahydrochloride Sigma) in TrisHCl 0.05 M, pH 7.6. Sections were
washed 4 times with PBS pH 7.5, and the reaction was stopped
by dehydration. Slides were stained with hematoxylin-eosin and
mounted in synthetic resin (Entellan) for light microscopy
analysis. The negative control was treated in the same way,
except that normal rabbit serum was used instead of anti-VTG
serum. Dark brown stains identified positive results.

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tions were performed using DH5 competent cells (Gibco


Invitrogen). Positive clones were sequenced using LICOR IR2
DNA sequencer. As internal control of RNA integrity and cDNA
quality, the 18s rRNA gene (Forward 5-GTT AAT TCC AGC
TCC AAT AGC GTA-3, Reverse 5-GAA CTA CGA CGG TAT
CTG ATC GTC-3) was amplified under the following PCR
conditions: one cycle at 94 C for 1.5 min, and 35 cycles at 94 C
for 1 min, 57 C for 1 min, and 72 C for 1 min, rendering a
product of 453 bp. To analyze gene expression, both genes were
PCR-amplified and visualized under UV light in a 2% agarose
gel in TrisAcetate (TAE) buffer stained with ethidium bromide,
digitized in an Epi Chemi II dark room using the Labworks
Imaging and Analysis software (UVP BioImaging Systems).
2.5. Morphological description of the follicles
The intact gonads were fixed in 4% formaldehyde (W/V) in
PBS pH 7.5 for 1 week and then paraffin-embedded using

2.4. VTG mRNA expression in maternal liver


Females in different ovarian developmental stages were
selected for this analysis. Using sterile blades, 3 liver samples
from each developmental stage were collected in RNAlater
(Ambion). Total RNA was isolated using Trizol reagent (Gibco
Invitrogen) followed by DNAse I treatment. cDNA synthesis
was performed at 45 C with 5 g of total RNA, Improm-II
reverse transcriptase (Promega), and random primers (Promega). PCR amplification for the vitellogenin gene of both
species was carried out with the following primers: VTGforward 5 AAR ACC TAT GTG TAC AAG TAT GAG G 3 and
VTG-reverse 5 GTC TTC TTG AKG TTR AGC TG 3 (where
K = G / T and R = A / G). PCR conditions were performed as
follows: one cycle at 94 C for 1.5 min, and 35 cycles at 94 C for
1 min, 50 C for 1 min, 72 C for 1 min, rendering a product of
350 bp. The identity of both gene fragments was confirmed by
DNA sequencing; briefly PCR products were purified and
ligated into a pGEM-T cloning vector (Promega). Transforma-

Fig. 1. Condition factor of females collected at both locations. a) Girardinichthys viviparus from Texcoco Lake (19 25 N; 98 55 W), Estado de
Mexico. b) Ameca splendens from Veneros de Teuchitln (spring from Ameca
river, 20 41 N; 103 43 W), Jalisco, Mexico. W = total mass (g); L = standard
length (mm).

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conventional methods (Ortiz-Ordez et al., in press). Serial 8micron-thick sections were obtained. The slides were then
stained with hematoxylineosin and mounted in synthetic resin
(Entellan) for light microscopy analysis.
3. Results
3.1. Condition factor and reproductive aspects
Gestating females were collected over a year period. A
higher condition factor was observed in the female black fin
goodeid (Fig. 1 a) compared to the female butterfly split-fin
goodeid (Fig. 1 b). Under controlled laboratory conditions,
reproductive information was recorded. In the butterfly split-fin
goodeid, the age of first maturation was 6 months, the gestation
period was 2 months, and the fish gave birth to an average of 21
live offspring with a 40% viability. In the black fin goodeid, the

Table 1
Vitellogenin levels (%) during gestation of Girardinichthys viviparus (Stage
2 = vitellogenesis. Stage 3 = beginning of intraluminal gestation. Stage
4 = embryonic growth. Stage 5 = prenatal embryos)
Target
organ

Stage 2

Stage 3

Stage 4

Stage 5

Parent
muscle
Embryo
Gonad
Liver

0.16 0.2

1.82 1.1

2.03 0.9

1.34 0.7

25.24 20.7 3.82 0.7


0.43 0.3
78.19 31.5 69.71 25.9 73.29 41.1 53.07 15.7
21.65 7.1
3.22 2.6 20.86 13.9 45.15 30.6

Standard deviation in percentage.


Statistical differences between developmental stages: p b 0.05. p b 0.01.
p b 0.001.

age of first maturation was 9 months, the gestation period was


2.5 months, and the fish gave birth to an average of 36 live
offspring with a 25% viability. In both species, viability improved in older females (N1 year old); the average viability
reached almost 85%. In black fin goodeid embryos, there was
an increased dry weight of 21,800% 300% related to the
mature oocyte.
3.2. VTG quantification during embryonic development
VTG was present during embryonic development of the black
fin goodeid (Fig. 2 a). Higher VTG concentrations were detected
during vitellogenesis in the maternal liver and gonad. Subsequently, gonadal VTG concentration decreased until the end of
gestation, whereas in the liver, the VTG concentration increased
in the last stage of gestation. In the maternal muscle and in the
embryos, the VTG concentration was lower than in other target
organs; the maximum VTG concentration in the muscle and in
the embryos was detected during early development (stage 3).
The distribution of VTG (%) is shown in Table 1.
In the butterfly split-fin goodeid, VTG concentrations in the
liver and in the gonad increased at the end of the gestation
period (Fig. 2 b), whereas in the muscle, the maximum concentration was detected during stage 3. In the embryos, higher
VTG concentrations were noted during the last two developmental stages. VTG was quantified mainly in the gonads, and

Table 2
Vitellogenin levels (%) during gestation of Ameca splendens (Stage
2 = vitellogenesis. Stage 3 = beginning of intraluminal gestation. Stage
4 = embryonic growth. Stage 5 = prenatal embryos)

Fig. 2. VTG quantification in goodeid fish by a hybrid ELISA in different


maternal organs and embryos, in each gonadic developmental stage. a) Girardinichthys viviparus. b) Ameca splendens. Significant differences p b 0.05;
p b 0.01; p b 0.001. Two-way ANOVA with a TukeyKramer test.

Target
organ

Stage 2

Stage 3

Stage 4

Stage 5

Parent
muscle
Embryo
Gonad
Liver

4.38 1.4

77.59 24.0
18.02 2.4

3.38 1.1

0.59 1.7

0.66 1.7

0.41 0.0 2.74 0.4 2.77 1.9


81.23 2.8 77.36 3.4 66.62 2.3
14.98 0.1 19.31 0.1 29.95 0.2

Standard deviation in percentage.


Statistical differences between developmental stages: p b 0.001.

A. Vega-Lpez et al. / Comparative Biochemistry and Physiology, Part A 147 (2007) 731742

the target organ distribution was similar to that seen in the black
fin goodeid (Table 2).
3.3. VTG detection by immunohistochemistry
The ovary of the black fin goodeid is composed of an
external serous wall of connective tissue and an internal
smooth muscle layer that is supplied by capillaries, as well as
connective tissue that supports the simple cuboidal epithelial
tissue that forms long and thin folds covering most of the
ovary lumen. Pigmented cells and blood vessels are observed
in the connective tissue. The cavity of the ovary is divided by
an ovarian septum that is covered by simple cuboidal
epithelium to which is attached connective tissue with
capillaries and smooth muscle fibers. Oocytes at different
developmental stages surrounded by follicular cells forming
the granulosa and theca interna layers are located towards the
ends of the septum between the connective tissue and the
folds. After fertilization, intrafollicular gestation begins and

735

continues until segmentation; subsequent development takes


place in the ovary (intraluminal gestation). At this time,
growing embryos, from stage 3 to stage 5 (close to birth), are
observed.
Studies have shown that, in the ovarian wall, specifically in
the serous layer and the smooth muscle fibers, VTG is present
during different developmental states (Fig. 3 b), as well as in
the ovarian stroma at stage 2 (Fig. 3 c and d). The presence of
VTG was also observed in the oocyte membrane during
vitellogenesis (Fig. 3 f). In the septum, VTG was detected in
the simple cubical epithelium during gestation (Fig. 3 g). In
the trophotaeniae region, VTG was observed on the surface of
the flat and simple columnar epithelia (Figs. 3 h, i, 4 b) during
intraluminal gestation (stages 3 to 5). In stage 3 to 5 embryos,
VTG was observed in the dermoplacenta region (where the
placenta makes contact with the wall of the ovary, Fig. 4 c, d,
e, f), as well as in the finfold (Fig. 4 e and h) and the body of
the embryo (Fig. 4 f). Finally, in the prenatal embryo, VTG
was detected in the head (Fig. 4 i).

Fig. 3. VTG detection in histological sections by immunohistochemistry during embryonic development of Girardinichthys viviparus. (a) External serous wall, stage
2, control (40) (b) External serous wall, stage 2, VTG-positive (40). (c) Ovarian stroma, stage 2, VTG-positive (40). (d) Spongy ovarian stroma, stage 2, VTGpositive (100). (e) Vitellogenic oocytes (stage 2), control (40). (f) Vitellogenic oocytes (stage 2), VTG-positive (40). (g) Ovarian septum, stage 3, VTG-positive
(100). (h) Trophotaeniae insertion and embryonic muscle layers, stage 3, VTG-positive (40). (i) Transverse and longitudinal sections of trophotaeniae, stage 3,
VTG-positive (40). Arrows indicate the presence and location of VTG.

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Fig. 4. VTG detection in histological sections by immunohistochemistry during embryonic development of Girardinichthys viviparus. (a) Trophotaeniae, transversal
section, stage 3, control (100). (b) Trophotaeniae, transversal section, stage 3, VTG-positive (100). (c) Panoramic of fin folding and development of dermoplacenta
with the inner wall of the ovisac, stage 4 VTG-positive (10). (d) Panoramic of the dermoplacenta of the embryo's body with ovarian stroma, stage 4 (10). (e) Fin
folding, stage 4, VTG-positive and dermoplacenta (40). (f) Body of the embryo and dermoplacenta, stage 4 control. (g) Finfold, stage 5, control (40). (h) Finfold,
stage 5, VTG-positive. (i) Head of the embryo prior to birth, VTG-positive (placentotrophy) (10). Arrows indicate the presence and location of VTG. White squares
indicate the dermoplacenta.

3.4. VTG mRNA expression in the maternal liver based on


RT-PCR
This analysis revealed that different VTG expression
patterns were present in each species. In the black fin goodeid,
VTG was expressed during vitellogenesis (stage 2) (Fig. 5
right), whereas in the butterfly split-fin goodeid VTG was
expressed during intraluminal gestation (stages 3, 4, and 5)
(Fig. 5 left). Nucleotide sequences of 344 and 366 bp were
obtained for G. viviparus (GenBank AY845859), and A.
splendens (GenBank AY845860) cDNA fragments respectively. Both sequences correspond to the 5 region of the full-length
VTG mRNA.
3.5. Morphological description of the follicle
In general, it was observed that a single layer of cuboidal
granulosa cells in the butterfly split-fin goodeid and squamous

cells in the black fin goodeid follicle surrounded the growing


oocytes. The mature oocytes of both species had a single layer
of cuboidal granulosa cells. The main difference in the follicle
between the two species was related to the theca cells. In the
follicle of the black fin goodeid, very profuse theca cells were

Fig. 5. VTG mRNA expression in maternal liver at different gonadic


developmental stages analyzed by RT-PCR. Left) Ameca splendens. Right) Girardinichthys viviparus. 2) Vitellogenesis and intrafollicular gestation. 3) Beginning
of intraluminal gestation. 4) Embryonic growth. 5) Prenatal embryos. +) Positive
control (cloned VTG fragment). ) Negative control (ddH2O). M = molecular
weight marker (phi-X/HaeIII).

A. Vega-Lpez et al. / Comparative Biochemistry and Physiology, Part A 147 (2007) 731742

Fig. 6. Girardinichthys viviparus and Ameca splendens follicles. a) Growth oocyte


of A. splendens (100). b) Mature oocyte of A. splendens (100). c) Growth oocyte
of G. viviparus (100). d) Mature oocyte of G. viviparus (100). TICs= theca
interna cells. GCs= granulosa cells. Ov = Oocyte.

observed, which were surrounded by a compact stroma (Fig. 6).


In contrast, in the butterfly split-fin goodeid, the granulosa cells
were surrounded only by a few, thin theca cells, and the stroma
had a very lax appearance.
4. Discussion
This study demonstrated for the first time that VTG is
present during goodeid fish gestation, and that it also functions
as a nutrient supplier. The earliest recorded evidence relating to
the role of VTG in embryonic development in this fish family
was presented by Blake in 1867, who observed proteins, lipids,
phosphates, and iron in the ovarian fluid of goodeid fish such as
Neotoca spp. (Mendoza, 1940). Arukwe and Goksyr (2003)
considered VTG and the zona radiata proteins to be some of the
oldest proteins that are linked to oocyte development, since they
can be found from insects to some eutherians, such as
marsupials and monotremes (Rothchild, 2003). It is generally
believed that the role of VTG is limited to oocyte yolk
formation. However, we believe that VTG has other adaptive
and evolutionary implications. Therefore, we will focus our
discussion on three major points.
4.1. VTG distribution patterns were similar in both species and
reflect the importance of this biomolecule in intraluminal gestation
VTG distribution in both species revealed that the gonads
(ovisac, stroma, and septum) are the immediate target organ.
Several authors have demonstrated that VTG is synthesized in
the liver, released into the blood stream, reaches the gonads, and
binds to specific receptors on the oocyte surface (Rothchild,

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2003). VTG is then unfolded by cathepsin D to produce yolk


proteins (lipovitellin I, II, and phosphovitellin) in oviparous,
lecitotrophic (ovoviviparous), and matrotrophic (viviparous)
fish (Nicolas, 1999; Arukwe and Goksyr, 2003).
In both species, the maternal liver had the second most
elevated concentration of VTG; based on the literature, one
would expect that the liver VTG concentration would be more
elevated, since the liver is the site of VTG synthesis. However,
the liver may not store this protein; therefore, liver VTG concentrations may only be the result of the synthetic activity
during embryonic development. In contrast, Wourms et al.
(1988) reported that the liver of cartilaginous fish could, under
certain circumstances, function as a VTG storage organ.
VTG is transported in the blood stream (Nicolas, 1999;
Arukwe and Goksyr, 2003). In both species, the VTG concentration in the maternal muscle was low compared to other target
organs (liver and gonad). It is possible that muscle VTG levels
are the result of maternal hematic circulation. Unfortunately,
these phenomena have not been documented in goodeid fish or
in other fish with matrotrophic viviparity, but have only been
inferred in previous reports (Turner, 1940b; Wourms et al.,
1988).
During different developmental stages, major differences in
VTG distribution were observed in the embryos of both species.
In the black fin goodeid, the highest VTG concentration was
detected at the beginning of intraluminal gestation (stage 3),
corresponding to the maximum amount of VTG in the maternal
muscle, suggesting that an early supply of VTG occurs at the
beginning of gestation regardless of the presence of pre-existing
yolk. Turner (1940b) mentioned that during early intraluminal
gestation, different goodeid fish embryos, such as Ataenobios
toweri, Goodea atripinnis (luitpoldii), Characodon lateralis,
and Girarnichthys multiradiatus (Lermichthys multiradiatus),
with residual yolk from the intrafollicular gestation move to the
ovarian lumen. This observation does not conflict with our
results, since the VTG concentration in the embryos was
determined in the homogenate of the whole body, and it is
possible to find cross-reactivity of polyclonal anti-VTG
antibodies with yolk proteins, since the antibodies recognize
conserved regions of the protein or its polypeptides (VegaLpez et al., 2006).
In the butterfly split-fin goodeid embryos, the VTG concentration increased with embryonic development. This increase was related to the VTG level in the maternal liver. The
differences observed between the two species are probably due
to a different motherembryos relationship with respect to the
supply of VTG for embryonic development. However, no
previous studies have demonstrated that this protein is synthesized during intraluminal gestation as a nutritional source for
the embryos.
Using immunohistochemical techniques, the present study
demonstrated that the ovary of the black fin goodeid is able to
transport exogenous, large proteins (such as VTG) during different embryonic developmental states. The existence of
special zones that are associated with the hemotropic transit
of VTG towards the gonad could be proposed. The massive
uptake of this protein seems to be more related to localized

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extravasation in some regions of the serous and the muscular


layer of the ovisac (Fig. 3 b) than to cellular level processes.
Previous studies have reported that hemotropic transfer of
metabolites during intraluminal gestation could be the dominant
mechanism for histotrophe formation in Zoarces viviparus and
Jenynsia lineata (Wourms et al., 1988). Additionally, we
observed that VTG uptake occurs during the whole period of
intraluminal gestation in goodeids. It is important to remember
that some nutritional supplies in the ovarian lumen could
originate in the follicle (De Vlaming et al., 1983). Lombardi and
Wourms (1985a) detected 4 or 5 types of proteins in the ovarian
fluid of the butterfly split-fin goodeid, and they proposed that
these proteins have a maternal origin. However, we believe that
at least some of these proteins could be VTG polypeptides.
Previous studies have demonstrated the existence of 7
polypeptide chains in the VTG protein of the black fin goodeid
(Vega-Lpez et al., 2006), as well as in the butterfly split-fin
goodeid (unpublished data). Therefore, even though an
electrophoretic analysis of the proteins or polypeptides of the
ovarian fluid was not done, the existence of VTG in the gonad
was shown on immunohistochemistry. This does not imply that
VTG is the main nutrient supplier, since VTG represents a small
fraction of total proteins.
During vitellogenesis, VTG is stored in the spongy
connective tissue of the ovarian stroma; in goodeid fish, it has
been reported that the ovarian stroma becomes spongy during
vitellogenesis and fertilization (Turner, 1933, 1940b; Mendoza,
1940; Miller and Fitzsimons, 1971; Daz-Pardo and OrtizJimnez, 1985) due to follicular cells acquiring secretory
properties necessary for intrafollicular gestation (Turner, 1940b;
Wourms et al., 1988). We observed that the stroma spongy
tissue probably functions as a VTG reservoir for developing
eggs (Fig. 3 c y d), regardless of the secretory properties of the
follicular cells, given that in later stages of intraluminal
gestation these cells disappear, leaving behind small portions
of stroma associated with the dermoplacenta (Fig. 4 e).
In the vitellogenic phase, VTG was detected only in the
perivitelline space of the oocyte (Fig. 3 f); Rothchild (2003)
suggested that VTG passes through capillary vessels from theca
interna cells (TICs) to reach the basal membrane of the
granulosa cells (GCs), and then to finally be deposited in the
perivitelline space where it binds to specific receptors on the
oocyte surface. GCs are crucial for VTG uptake and function
under the influence of gonadotrophic hormones (GtH). GtH I
increase GC permeability for the transit of VTG towards the
oocyte (Nicolas, 1999; Arukwe and Goksyr, 2003; Rothchild,
2003) and stimulate estradiol (E2) synthesis (Kagawa et al.,
1982). It is likely that the fine transit of VTG between TICs,
capillaries, and GCs could be observed through antigenantibody reactions using ultrastructural techniques.
The present investigation has demonstrated for the first time
that VTG uptake occurs through the finfolds (hypertrophy of the
middle region of the caudal fin), since VTG was detected in the
body of the embryo during intraluminal gestation (Fig. 4 e, h).
Mendoza (1958) suggested that this embryonic organ has a role
in gas exchange and, probably, in nutrient exchange in Goodea
atripinnis (luitpoldii) embryos. The embryonic surface in-

creases up to 60% by hypertrophy of the caudal fin, which is


related to ovigerous folds (Wourms et al., 1988). The existence
of a dermotrophic placenta is suggested by the strong relationship of the finfolds with the ovarian wall and the finfolds of
the black fin goodeid (Fig. 4 c). A dermotrophic placenta has
two main functions: gas exchange due to its pronounced
vascularization, and nutrient absorption. However, scanning
electron microscopy has revealed that embryo finfolds of the
embiotocid fish Micrometrus minimus are rich in microplicae
and lack microvilli; therefore, the role of the dermotrophic
placenta may be limited to gas exchange (Wourms et al., 1988).
Wourms and Lombardi (1985) found that spatula-shaped
extensions of the fins known as epaulettes in Rhacochilus
vacca embryos have endocytic activity for small molecules,
such as amino acids and sugars. In contrast, the results of the
present study showed that the finfolds of the black fin goodeid
are able to absorb complex nutrients, such as VTG, probably
through extravasation mechanisms related to the dermoplacenta
(Fig. 4 e, f). Nevertheless, finfolds may lack the lytic capacity of
trophotaeniae, given that VTG digestion takes place in the
embryo's body.
Transverse sections of the trophotaeniae of the black fin
goodeid embryos revealed the diffuse presence of VTG, which
involved the main blood vessel of this organ. This is undeniable
evidence of the transport of VTG to the final target, which, in
goodeid fish, is related to embryo nutrition (Fig. 3 h y i). The
importance of trophotaeniae apical cells in nutrient absorption
and digestion through endocytosis-associated microvilli, in the
epithelial transept transport (Fig. 4 b) related to the formation of
a trophotaenial placenta (Fig. 3 h), and its role in gas exchange
(Lombardi and Wourms, 1985b) have been reported. The
presence of receptors in trophotaenial cells of the butterfly splitfin goodeid, which regulate endocytosis of lysine-rich proteins
(Schindler, 2003a) and aminopeptidases, which function as
scavenger receptors to mediate endocytosis (Schindler, 2003b),
has been noted. Using histochemical techniques, alkaline and
acid phosphatases, which reflect lysosomal enzymatic activity,
have been detected in the trophotaeniae of the butterfly split-fin
goodeid (Lombardi and Wourms, 1985b). According to our
observations, VTG hydrolysis occurs during its transit through
the lysosomes, and the diffuse VTG presence (Fig. 3 h y i) that
was noted represents non-hydrolyzed VTG that remains after
VTG lytic activity in the trophotaenial cells. Nevertheless, it
was not possible to completely verify that VTG uptake occurs
through the connective tissue towards the capillaries of the
trophotaeniae. In contrast, Schindler and de Vries (1986) reported that the trophotaeniae of the black fin goodeid is able to
take only small molecules; it is possible that these different
findings are due to differences in the techniques that were used
in the studies.
VTG was observed in the head of the prenatal black fin
goodeid embryos (Fig. 4 i), which is evidence of placentotrophic nutrition (Wourms et al., 1988). Under these circumstances, the presence of VTG may be due to ovarian fluid
suction. Boechlert and Yoklavich (1984) have shown that, as a
consequence of peristaltic contractions, prenatal rockfish
(Sebastes schlegeli) embryos had opaque material in the

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hindgut. In the embryonic intestine of viviparous fish, nutrient


absorption apparently involves the transport or diffusion of
small molecules, or endocytosis of macromolecules, especially
proteins (Wourms et al., 1988). Our results do not suggest that
intracellular digestion of VTG occurs in the intestine; however,
they could reflect the importance of placentotrophy as a trophic
adaptation of the embryo, at least at the end of gestation. It is
possible that such a strategy has been invested in reproduction
as a means of energy conservation (Wourms et al., 1988).
4.2. VTG gene expression occurred at different times during
gonadal development of the two species, which could reflect
different life strategies/histories of the species
The amount of VTG synthesized by the female black fin
goodeid was one order of magnitude higher than that
synthesized in the female butterfly split-fin goodeid (Fig. 2 a
y b). The weight gain of the black fin goodeid embryo by the
time of birth was 21,800%. Lombardi and Wourms (1985b)
found that the butterfly split-fin goodeid embryos increased in
dry weight up to 15,000% compared to the oocytes. These
observations suggest a direct relationship between the amount
of VTG synthesized during embryonic development and the
gain in dry weight. To confirm these findings, similar studies
should be performed in other goodeid fish in which the highest
and lowest increases in dry weight have been detected, such as
Zoogoneticus quitzeoensis (38,700%) and Goodea atripinnis
(1100%), respectively (Wourms et al., 1988; Hollenberg and
Wourms, 1995).
Furthermore, in both species examined in this study, the
amount of VTG synthesized was related to the condition factor,
which was higher in the black fin goodeid (Fig. 1 a). The
condition factor is closely related to food availability in
lecitotrophic fish females, since fat reserves depend on the
amount of VTG synthesized and, therefore, on the amount of
yolk that is present (Wourms et al., 1988). Similarly, it has been
observed that in fish with matrotrophic viviparity, such as Heterandria formosa, the amount of food intake that occurs during
gestation is related to maternal reserves (Wourms et al., 1988).
Based on our data, fish with a higher condition factor had higher
fat deposits, and, consequently, greater amounts of VTG were
synthesized during gestation.
In the two species, VTG synthesis occurred at different times
during gonadal development; this may reflect that goodeid fish
have different life strategies/histories. Reproductive characteristics are able to reveal differences in life strategies/histories of
different species; they depend on genetic plasticity that allows
the species to reach reproductive fitness in a particular environment (Mann et al., 1984). In the black fin goodeid, peak VTG
mRNA expression occurred during vitellogenesis (Fig. 5 right),
though the protein was detected throughout gestation. This
result suggests that VTG is stored, probably in the fat tissues of
the abdominal cavity, through affinity of the prosthetic lipidic
group of VTG. Fat deposits have been reported in mature
females during vitellogenesis (Arukwe and Goksyr, 2003), and
are probably generated by VTG accumulation in matrotrophic
fish species, such as the female black fin goodeid. Korsgaard

739

and Petersen (1979) demonstrated that lipids are among the


most abundant nutrients found in ovarian fluid of gestating
blenny (Z. viviparus) females, and that they are synthesized in
the liver in response to E2 levels. Subsequently, they are transported by the maternal blood stream to the gonads and the
ovarian fluid. Davis (1997) suggested that lipoprotein fat
transport is probably one of the most significant functions that
allows the survival and reproduction of eukaryotes. Our observations, which included maximum VTG synthesis, VTG
storage in fat tissues, and VTG release during gestation of the
female black fin goodeid, support this suggestion.
In contrast, VTG mRNA expression in the female butterfly
split-fin goodeid occurred during intraluminal gestation (Fig. 5
left) and was related to the fish's lower condition factor. It has
been demonstrated that some goodeid species are able to mature
eggs for the next brood even during embryonic development of
the current brood (Wourms et al., 1988). The butterfly split-fin
goodeid oocytes reach the vitellogenic stage at the beginning of
the previous intraluminal gestation. The fact that the female is
naturally synthesizing VTG during gestation has not been previously noted. Moreover, we observed superfetation in butterfly
split-fin goodeid females in reservoirs when an increased food
supply was available (unpublished data). However, it is
important to remember that lecitotrophic fish females, such as
the eelpout (Z. viviparus), are able to synthesize VTG during
embryonic development under artificial stimuli (e.g. presence of
E2 or xenoestrogens) (Rasmussen et al., 2002); this has also been
shown indirectly by studying the uptake of phosphatidylcholine
in the yellowtail rockfish (MacFarlane and Bowers, 1995).
The present study suggests that various goodeid fish have
different life strategies/histories. Some previous reports that are
not directly related to the current study support this idea. For
instance, Webb (1998) and Webb et al. (2004) reported that G.
viviparus belongs to the Girardinichthyini tribe, whereas A.
splendens belongs to the Chapalichthyini tribe, and that both
tribes are perfectly differentiated. Ritchie et al. (2005)
commented that the innovation of matrotrophic viviparity was
a key factor in the radiation of this group, together with
biogeographic variance. In addition to studying other goodeid
fish, it would be interesting to perform comparative studies
similar to this investigation among different species from the
Poeciliidae, Zoarcidae, Anablepideae, Embiotocidae, and
Fundulidae families (Turner, 1940b; Wourms et al., 1988;
Webb, 1998). Even though both species present different life
strategies/histories, VTG synthesis during embryonic development of this family could probably help explain the transition
between lecitotrophy and matrotrophic viviparity.
4.3. VTG supply during embryonic development of viviparous
fish is probably an adaptive strategy for the transition from
lecitotrophy to matrotrophic viviparity
It has been mentioned that the transition from oviparity to
viviparity in fish involves significant changes in the egg, the
embryo, and the mother (Wourms et al., 1988). The evidence
presented in our paper is consistent with previous reports, which
focused on the central issue of the transition between

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lecitotrophy and matrotrophic viviparity in bony fish with


intraluminal gestation. We believe that two key events are
involved in this transition:
a) Gradual loss of the ability to produce VTG. This is one of the
earliest mechanisms that was proposed to explain the
evolution of viviparity in goodeid fish (Turner, 1933; Turner,
1940b). Initially, the evidence was based on oocyte size and
not on VTG expression/quantification. Depending on the
amount of VTG synthesized, as well as on the pattern of VTG
synthesis, one could speculate that the black fin goodeid is
older than the butterfly split-fin goodeid, since lower amounts
of VTG are associated with more evolved species (Turner,
1940b; Wourms et al., 1988). The black fin goodeid
synthesizes a larger amount of VTG than the butterfly splitfin goodeid, and its VTG synthesis is limited to the
vitellogenic period, which occurs in oviparous (Nagler and
Idler, 1990; Parks et al., 1999; Bowman et al., 2000; Celius
et al., 2000; Lattier et al., 2001, 2002) and ovoviviparous
(Korsgaard and Andersen, 1985; MacFarlane and Bowers,
1995) species. In the same way, VTG accumulation outside
the oocyte could be a transition strategy towards a more
evolved fish species. The ability of the black fin goodeid to
store VTG in fat tissues may be considered to be a reproductive strategy that is evolutionarily equivalent to yolk
accumulation in oviparous and lecitotrophic fish. In contrast,
in females of the butterfly split-fin goodeid, VTG synthesis
occurred continuously during intraluminal gestation. VTG
was synthesized in lower amounts than in the female black fin
goodeid, which is a clear indication of the gradual loss of the
ability to produce VTG. Other investigations using different
criteria support the geological and evolutive age of the black
fin goodeid and the butterfly split-fin goodeid. Webb (1998)
and Webb et al. (2004) studied a partial sequence of subunit I
of the mitochondrial gene cytochrome oxidase and demonstrated that both species are monophyletic; the black fin
goodeid originated in the late Miocene, whereas the butterfly
split-fin goodeid originated in the Pliocene. On the other
hand, Riehl and Greven (1993) investigated the zona radiata
of the black fin goodeid oocyte and found that it was
composed of two differentiated layers (inner and outer
layers), while the butterfly split-fin goodeid oocyte was
composed of one homogeneous layer. Zona radiata layer
reduction is important in viviparity evolution (Guillette,
1989), since species in transition have a reduced egg shell
thickness (Wourms et al., 1988). Oocyte protection barriers
against the environment (outer layer of the zona radiata) are
unnecessary in intraluminal or intrafollicular gestation. These
phenomena have been observed in oviparous and in lessevolved viviparous bony fish (Poeciliidae, Hemirhamphide,
Zoarcideae), in which the zona radiata is present as two, thick,
well differentiated layers (Wourms et al., 1988; Riehl and
Greven, 1993). Together, these reports support the patterns of
VTG synthesis found in the current study.
b) Changes in the symbiotic relationships between the oocyte
and follicular cells. The main difference between the two
goodeid fish species was found in the follicle. The follicles

differed with respect to the number and thickness of the theca


interna cells (TICs), as well as their association with the
granulosa cells (GCs) and the surrounding stroma. In the
black fin goodeid, the TICs were larger, and a greater
quantity of VTG was synthesized during gestation. It is
possible that the TICs and their interrelation with the GCs
resulted in greater VTG synthesis. Rothchild (2003) has
documented that oocyte maturation depends on GCs, and
that GCs depend on oocyte maturation to proliferate and
differentiate. The proliferation and differentiation of GCs, as
well as their relationship with TICs, facilitates the optimum
balance of estrogen levels needed to maintain VTG production (Nicolas, 1999; Arukwe and Goksyr, 2003; Rothchild, 2003). This situation also favors the conversion of
androgens into estrogens through aromatase action (Kagawa
et al., 1982; Wourms et al., 1988). In turn, E2 can affect the
pituitary gland as a negative feedback mechanism (Arukwe
and Goksyr, 2003). It has been proposed that, once the
optimum size has been genetically determined, unknown
factors allow oocyte VTG receptor saturation (Tata and
Smith, 1979; Arukwe and Goksyr, 2003; Rothchild, 2003).
As well, there may be physical limitations to oocyte size
(Wourms et al., 1988). It is possible that the relationship
between the oocyte and the GCs plays a role in the transition
from lecitotrophic to matrotrophic viviparous fish; this can
occur through modifications in the number of TICs and their
relationships with the GCs, since follicle cells' symbiotic
relationships have been associated with the plasma E2 levels
and/or a decrease in the number of oocyte VTG receptors.
This topic requires further research.
Due to the changes in the relationship between oocyte and
follicular cells, together with a gradual loss of the ability to
produce VTG, there have been changes in the trophic
relationships between mother and embryo in fish with
matrotrophic viviparity. This has been extensively documented
(Wourms et al., 1988), and further demonstrated in the present
study. Wourms et al. (1988) also reported on several aspects of
the transition from ovoviviparity to matrotrophic viviparity,
including internal fertilization, sexual dimorphism, prenuptial
courtship, the retention of the fertilized egg inside the body of
the mother, the loss of autonomy of the embryo (due to yolk
loss), development of the genital tract, the development of a
neuroendocrine control system, and immunological relationships between the mother and embryo. However, these characteristics are shared and are probably similar in lecitotrophic
and matrotrophic fish. The result of reproductive strategy
changes was less-numerous but more mature offspring
(Wourms et al., 1988; Rothchild, 2003). This was observed
in the black fin goodeid (36 livebearer, tending to an Rstrategy) and the butterfly split-fin goodeid (21 livebearer,
tending to a K-strategy). Rothchild (2003) mentioned that
evolution of eutherian viviparity was the result of oocyte
fragility, follicle instability, and the loss of the ability to
produce VTG, due to a quiet proliferation of homozygous
populations that was unable to produce VTG inside a
heterozygous population.

A. Vega-Lpez et al. / Comparative Biochemistry and Physiology, Part A 147 (2007) 731742

Based on our data, VTG participates in the nutrition of


goodeid fish, though its role is not limited to yolk production.
G. viviparus is older than A. splendens since it produces more
VTG, which is directly related to the weight gain of the embryos
at the time of birth and to the mother's condition factor. VTG is
synthesized during the vitellogenic period; it is probably stored
in the mother's fat tissues and is released during intraluminal
gestation. The butterfly split-fin goodeid produced less VTG,
which is related to less weight gain of the embryos at the time of
birth and the lower condition factor of the mother, as well as
continuous VTG synthesis during intraluminal gestation that
was related to the development of new mature oocytes. The
evolution of matrotrophic viviparity involved a gradual loss of
the ability to synthesize VTG; this loss was compensated by a
continuous but lower synthesis of VTG during the gestation
period.
Acknowledgments
We are grateful to SEMARNAT, Direccin General de Vida
Silvestre (Mxico) for authorizing the collection of parent
specimens and most particularly to biologist Gabriel Solano and
Dolores Morales. Our thanks also to the staff of the Lago de
Texcoco project, CNA, in particular MVZ Fernando Nez for
the support in the field. This study was financed by the Instituto
Politcnico Nacional, CGPI code 20041154. Ethel GarcaLatorre and Maria Lilia Domnguez-Lpez are fellows of
COFAA-IPN, EDI and SNI.
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