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JOURNAL OF EXPERIMENTAL ZOOLOGY 284:575–585 (1999)
Alterations in Serum Steroid Concentrations in the
Clearnose Skate, Raja eglanteria: Correlations With
Season and Reproductive Status
L.E.L. RASMUSSEN,1* D.L. HESS,2 AND C.A. LUER3
Department of Biochemistry and Molecular Biology, Oregon Graduate
Institute of Science and Technology, Portland, Oregon 97291-1000
2
Department of Reproductive Science, Oregon Regional Primate Research
Center, Beaverton, Oregon 97006
3
Mote Marine Laboratory, Sarasota, Florida 34236
1
ABSTRACT
Serum steroid hormones in the peripheral circulation of the clearnose skate, Raja
eglanteria, were measured at the time of capture and at various times throughout the year while
the animals were maintained as a captive breeding population. These analyses demonstrate interesting correlations between changes in hormone concentrations and annual reproductive events.
Animals were sampled once (78 females, 20 males) or multiple times (15 females). For both groups
of females, 17β-estradiol was detected throughout the year with significant elevations occurring
during October and November when ovarian follicles begin to mature (as determined through
necropsy examinations), and January and February when maximum mating activity is observed
and egg laying begins. Testosterone and dihydrotestosterone concentrations were significantly elevated in females only during January and February. Testosterone elevations were synchronous
with longer-term elevations in 17β-estradiol in females sampled either once or repetitively. Testosterone concentrations in males were significantly elevated during times of maximum breeding
activity compared to periods of sexual inactivity. Data from females sampled during five stages of
the egg laying process, as defined by the position of palpable egg capsules within the reproductive
tract, revealed that 17β-estradiol was highest when egg capsules were forming in the nidamental
gland (stage 2) or uterus (stage 3); testosterone and dihydrotestosterone were maximal when eggs
were in the uterus (stage 3) or cloaca (stage 4); and progesterone was significantly elevated immediately after oviposition (stage 5), suggesting a possible role for progesterone in the regulation of
sequential laying of egg pairs. J. Exp. Zool. 284:575–585, 1999. © 1999 Wiley-Liss, Inc.
A series of diverse reproductive strategies has
been developed by elasmobranch fish (sharks,
skates, and rays) during their long evolutionary
history, and the associated patterns of serum steroid hormone concentrations are as heterogeneous
as their varying morphologies and life histories.
Such complex reproductive adaptations include
oviparity (egg laying), aplacental viviparity (lecithotropic and histotropic live bearing), and placental viviparity (Wourms, ’77). The distinctive,
accompanying hormonal events are often as complex as hormonal cycles described during mammalian reproduction. This is especially evident in
recent endocrinological studies of placental elasmobranchs (Rasmussen and Murru, ’92; Rasmussen and Gruber, ’93; Manire et al., ’95; Manire
and Rasmussen, ’97). For example, in the bonnethead shark, Sphyrna tiburo, high serum concentrations of 17β-estradiol and testosterone are
© 1999 WILEY-LISS, INC.
evident during mating and preovulatory stages,
and significantly elevated progesterone is evident
during preovulatory, ovulatory, and postovulatory
stages; dihydrotestosterone (DHT) is only elevated
during the preovulatory period (Manire et al., ’95).
In non-placental elasmobranchs, such as the
aplacental viviparous electric ray, Torpedo marmorata (Lupo di Prisco et al., ’67), spiny dogfish,
Squalus acanthias (Callard et al., ’93), and the
oviparous little skate, Raja erinacea (Koob et al.,
’86), hormonal patterns are different. In these spe-
Grant sponsor: Biosphere Research Corporation; Grant sponsor:
Mote Marine Laboratory; Grant sponsor: The Disney Wildlife Conservation Fund.
*Correspondence to: L.E.L. Rasmussen, Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and
Technology, P.O. Box 91000, Portland, OR 97291-1000. E-mail:
betsr@bmb.ogi.edu
576
L.E.L. RASMUSSEN ET AL.
cies, evidence implicates gonadal hormones in the
direct regulation of the reproductive tract (Sumpter and Dodd, ’79; Dodd and Sumpter, ’84; Callard
and Klosterman, ’87; Callard et al., ’89; Koob and
Callard, ’91). For example, in R. erinacea, a species in which females undergo two reproductive
cycles each year during which egg pairs are laid
every five to seven days (Fitz and Daiber, ’63;
Richards et al., ’63), clear hormonal patterns establish seasonal changes in reproductive activity
in the populations studied. Two periods have been
observed wherein a higher proportion (60%) of females is reproductively active, although the population as a whole is reproductively active all year.
Elevated 17β-estradiol and testosterone are characteristic of the follicular phase, with the source
of hormones identified as the developing ovarian
follicles (Koob et al., ’86). Increases in serum progesterone in the little skate occur predominantly
in pre- to periovulatory periods in contrast to the
longer periods of elevation in mammals and placental sharks (Manire et al., ’95). Recently,
Callard et al. (’95) suggested that steroid hormones such as progesterone and 17β-estradiol act
as key gating mechanisms, controlling such
events as mate selection and mating, follicular
development and regression, steroidogenesis, development and maintenance of the reproductive
tract, ovulation, vitellogenesis, corpora lutea
events, and parturition or oviposition. In contrast
to R. erinacea, R. eglanteria is not reproductively
active year-round, with its year clearly divided
into a reproductive period and a period of reproductive quiescence. With nonoverlapping cycles,
the ability to associate hormonal changes with
season and reproductive status should be less
complicated than in R. erinacea.
The reproductive season for R. eglanteria appears to be related to temperature, since breeding activity for R. eglanteria in the Delaware Bay
(Fitz and Daiber, ’63) and winter breeding activity for R. eglanteria from the eastern Gulf of
Mexico (Luer and Gilbert, ’85) occur at the same
water temperature. Although Fitz and Daiber
(’63) never observed mating or laying of eggs, their
assessment of reproductive activity was based on
size–frequency distribution of ovarian eggs. Interestingly, Fitz and Daiber (’63) found two groups
of ovarian eggs, a reservoir group and a maturing group. The maturing eggs were assumed to
be for the ensuing reproductive season, while the
reservoir eggs were thought to make up the maturing group for the following year’s reproductive
activity. In addition, they describe the initiation
of the egg case formation in the shell gland prior
to ovulation (Fitz and Daiber, ’63).
Since 1981, the clearnose skate, R. eglanteria,
has been successfully bred and maintained in
captivity at Mote Marine Laboratory (Luer and
Gilbert, ’85; Luer, ’89). Under the appropriate
captive conditions, adult female clearnose skates
breed readily with adult males and begin to lay
fertile eggs (Luer and Gilbert, ’85; Luer, ’89). The
resulting embryos and laboratory-raised offspring have been utilized as a unique vertebrate
model for a variety of anatomical (Conrad et al.,
’94), physiological (Goldstein et al., ’93), biochemical (Bodine et al., ’89), and immunological
investigations (Rast et al., ’97). Because considerable information is available about the biology and behavior of reproduction in the clearnose
skate, and because the entire reproductive cycle
can be monitored in captivity, this species provides an excellent opportunity to decipher some
of the sequential and interrelated hormonal
changes occurring during the complex reproductive processes of follicular maturation, mating,
ovulation, fertilization, oviposition, and sexual
quiescence.
The reproductive period for R. eglanteria from
the eastern Gulf of Mexico is defined as beginning in late fall (October/November) when sexually mature individuals begin to inhabit the
near-shore waters. Based on necropsy examinations, this time period appears to be associated
with the development and ripening of ovarian
follicles for the ensuing reproductive season
(Luer, unpublished). Mating behavior occurs
from December through late March to early
April, with maximal copulatory activity during
January and February (Luer and Gilbert, ’85;
Luer, ’89). As the temperature of near-shore waters begins to increase, adults move offshore by
late February to early March in search of deeper,
cooler water and are absent from local waters
until the next fall. The captive conditions described here provide the only way to observe the
annual reproductive cycle.
Following a successful breeding event, sperm
make their way up the female’s reproductive tract
to the paired nidamental glands, where sperm
storage is presumed to occur. This presumption
is based on the presence of sperm in nidamental
glands examined through tissue imprints and histological sections, with no visible evidence of
sperm ever reaching the oviducts (Luer, unpublished). As ova mature, they are ovulated and
travel down the oviducts to the nidamental
REPRODUCTIVE HORMONE CYCLES IN RAJA EGLANTERIA
glands, where fertilization and encapsulation take
place (Luer, ’89). Egg laying begins in January
and continues through June, occasionally extending into early to mid July if the females are maintained at 20°C (Luer, unpublished). Eggs are laid
in pairs, one from each side of the paired reproductive tract, at intervals averaging 4–5 days between laying of successive egg pairs (Luer and
Gilbert, ’85). Typically, the last few egg pairs laid
at the end of the season are infertile (Luer, unpublished). Time frames for various reproductive
activities or events are shown in Fig. 1.
The present study describes serum steroid hormone concentrations and hormonal cycles in reproductively active clearnose skates. Specifically,
serum 17β-estradiol, testosterone, DHT, and
progesterone levels were measured and correlated
with the behavior or reproductive event occurring
at the time of blood sampling. This study not only
increases our knowledge of steroid hormones as
they relate to the diversity of reproductive strategies in elasmobranchs, but also provides practical information for rapidly growing interests in
captive elasmobranch breeding programs.
MATERIALS AND METHODS
Research animals
Clearnose skates, R. eglanteria, were collected
in near-shore waters off Sarasota, Florida. The
577
majority of the animals were collected in the late
fall and early winter. Animals were held initially
in flow-through outdoor tanks at seasonally dependent temperatures, ranging from 14–22°C.
When flow-through water temperatures reached
22°C, animals were moved to recirculating indoor tanks maintained at 20°C. The special conditions during the study comparing hormone
levels of female skates housed with and without
males included maintaining daily temperature
records.
Because adult females were maintained as active egg layers for the production of embryos and
laboratory-raised offspring for a variety of collaborative research applications, animals were not
sacrificed as part of this study. Therefore, information and estimates about ovulation, encapsulation, and egg movement were determined by
relative locations of successive egg pairs within
the reproductive tracts of egg laying females.
Twice each day, the presence of egg capsules was
inferred from gentle palpation of the dorsal surface of females, often as part of a routine examination prior to obtaining blood samples. Egg
positions were assigned to a stage in the egg laying process: Stage 1 had no eggs present (this
group included both immediately pre-ovulatory
females and post-ovulatory (greater than 2 days)
females. Stage 2 had egg capsules forming in the
region of the nidamental gland (in the process of
Fig. 1. Time line of events occurring throughout the annual reproductive cycle in the clearnose skate.
578
L.E.L. RASMUSSEN ET AL.
encapsulation). The location of the ova is uncertain here because ovulation occurs after the egg
capsules are partly formed. Stage 3 had eggs in
the uterus (encapsulation complete). Stage 4 had
eggs in the cloaca, and stage 5 had eggs laid
within 2 days. Because animals were observed
several times each day, it was possible to determine the time of oviposition within a few hours.
Blood samples were obtained from animals that
had been in captivity from 2 weeks to 1 year.
Blood samples (1–2 ml) were obtained via caudal
venipuncture using a 23-gauge short-bevel needle.
Samples were allowed to clot and were centrifuged for 15 min at 1286g. Serum was aspirated
and stored at –20°C until thawed for analysis for
steroid concentrations by radioimmunoassay
(RIA). Single blood samples were obtained from
78 females and 20 males spanning five different
breeding and egg laying seasons. Multiple samples
were obtained from 15 females with sampling intervals ranging from every 24 hr for 1 week to
bimonthly for 8 months. Animals were bled at the
time of capture as well as at various times after
acclimation to captive conditions.
Radioimmunoassays
Serum concentrations of 17β-estradiol, testosterone, DHT, and progesterone were determined by
RIA after purification by chromatography on
Sephadex LH-20 microcolumns. Serum aliquots of
500 µl were extracted by shaking for 5 min with 5
ml of freshly opened or redistilled diethyl ether.
After freezing the aqueous phase in an ethanol/
dry ice bath, the organic phase was decanted and
brought to dryness under a stream of air. The dried
extracts were resolubilized and sequentially chromatographed on two different Sephadex LH columns. On the first column (1.0 g LH-20; elution
mixture: hexane:benzene:methanol, 62:20:13 v/v),
17β-estradiol was separated from estrone and all
neutral steroids (Resko et al., ’75). The neutral
fraction from the first column was applied to the
second column (2.5 g LH-20; elution mixture:
hexane:benzene:methanol, 85:15:5 v/v), and the
appropriate fractions for progesterone, DHT, and
testosterone were collected (Resko et al., ’80).
The purified steroids were then estimated by
RIA as described previously (Resko et al., ’75;
Hess et al., ’81).
Extraction and chromatographic losses were
monitored by adding known amounts of tritiated
authentic steroids to independent samples of
skate serum and processing these samples in
parallel with the unknown samples. Recoveries
following the chromatography were 75% for 17βestradiol, 88% for progesterone, and 72 and 70%
for DHT and testosterone, respectively. Water
blanks were also processed in parallel with the
unknown samples to provide estimates of solvent
blank methods (pg/ml) for each steroid as follows: 17β-estradiol, 3.2 ± 0.4; progesterone, 22 ±
3.0; DHT, 18.6 ± 9.8; and testosterone, 3.5 ± 1.9.
Reported values were corrected for both procedural losses and method blanks before correcting for aliquots assayed. Each sample was
diluted with 500 µl of ethanol after chromatography and assayed at different volumes ranging
from 5–200 µl. The reported values are the average concentration calculated from aliquots
whose values fell between the 5–95% binding
limits of the appropriate standard curve following linearization with a logit-log transformation.
Intra-assay coefficients of variation did not exceed 12% for these assays, and the sensitivity
limits for the steroids determined in this study
were 17β-estradiol, 5; progesterone, 10; DHT, 5;
and testosterone, 5 pg/tube, respectively. All four
steroid hormones demonstrated complete parallelism in the RIAs when compared to the standards, supporting our assumption that the
purification procedure eliminated any major interfering substances.
Statistics
For data from the 78 singly sampled females,
standard deviations and standard errors of
mean were calculated for sample sizes greater
than three when parametric tests were allowed
(i.e., data met the criteria for normality and
equal variance). Data were indicated as mean
± SEM. Students’ t-tests were utilized to determine significant differences between these data
sets at significance levels of P < 0.05 (95%).
Data that did not meet the parametric criteria,
i.e., data that consistently failed the KolmogorovSmirnov test of normality (non-normal distribution and/or unequal variance), were expressed as
medians (25–75 percentile respectively). Data
were analyzed by nonparametric tests including
the Mann-Whitney Rank Sum test or KruskalWallis one-way ANOVA on ranks (Sokal and
Rohlf, ’95). To isolate the group(s) which was significantly different, Dunn’s method for pairwise
multiple comparisons was employed (Dunn, ’64).
Significant correlations between testosterone and
17β-estradiol concentrations in serially sampled
skates were determined by Pearson product moment correlation.
REPRODUCTIVE HORMONE CYCLES IN RAJA EGLANTERIA
RESULTS
Seasonal variation in serum steroid
hormone concentrations in female skates
The following data are one-time samples obtained from individual animals after at least 2
weeks in captivity. Statistically significant elevations are indicated in comparison to the reproductively inactive months of July and August. Serum
17β-estradiol was detected in all individual adult
female skates throughout the entire year. Monthly
medians demonstrated significantly elevated 17βestradiol levels during two periods (September
through November, and January and February)
compared to the reproductively inactive months
of July and August (Fig. 2A). The reproductive
events that occur during the months demonstrating high serum 17β-estradiol include the development of ovarian follicles (ovarian recrudescence)
during October and November and the start of mating activity during January and February. During
March through late June, egg laying continues, but
copulatory activity gradually decreases through
early April (Fig. 1). In contrast, during the summer
months female skates are reproductively inactive.
Serum testosterone was detectable in all adult
female skates assayed (Fig. 2B). Median values (25–
75 percentile) of serum testosterone concentrations
were significantly elevated in January through April
compared to the period from July through December (Fig. 2B), whereas the apparent elevations during June were not significantly different.
DHT concentrations in serum were low (Fig. 2C).
At times the amount was below the level of detection, i.e., in May, September and December. Similar significant differences were observed for DHT
as noted with testosterone. January and February concentrations were significantly higher than
April–May and September–December (Fig. 2C).
Progesterone concentrations were generally low
(Fig. 2D). However, occasional transitory eleva-
Fig. 2. Serum steroid concentrations in female skates
throughout their annual reproductive cycle, depicted as
monthly medians (25–75 percentiles). Skate sample size = 3–
15/month. (A) Monthly medians of 17β-estradiol (significant
elevations September–November and January–February, compared to July and August); (B) monthly medians of testosterone (significant elevations January–April, compared to
July–December); (C) monthly medians of dihydrotestosterone
(January and February levels significantly higher than April–
May and September–December levels); (D) monthly medians
of progesterone (February medians significantly higher than
March, July–August, and December). Kruskal-Wallis one-way
ANOVA on ranks significant difference at P = 0.05.
579
580
L.E.L. RASMUSSEN ET AL.
tions from four-fold to 100-fold were observed.
Medians for progesterone concentrations during
February were significantly higher than during
three other periods: March, July–August, and December. July and August are reproductively inactive months; mid-December marks the initiation
of copulatory behavior for the season, and egg laying generally has not yet begun; whereas February is characterized by periods of copulation and,
in some years, maximum egg laying (Fig. 1). Although there was a 200-fold difference in absolute levels, P and T exhibited similar temporal
patterns, i.e., the levels were highest in January,
February, and April (Fig. 2B and D).
Comparison of serum steroid hormone
concentrations in male and female
adult skates
The comparison of serum steroid hormone concentrations in adult male (n = 20) and female (n
= 20) skates during one reproductive season
(1995) demonstrated several significant differences between the sexes (Table 1). The two sexes
were compared during two (December and January) of the three (December, January, and February) months when observed copulatory activity
was greatest. During those 2 months, it was possible to obtain equal numbers of blood samples
from males and females.
Concentrations of 17β-estradiol were significantly higher in females than males (670 ± 106.8
vs. 76.0 ± 44.9 pg/ml) during December. By January, female values had risen three-fold to 2480 ±
106.8 pg/ml (Table 1), whereas male levels had
decreased to 22.8 ± 7.8 pg/ml.
In males, the concentrations of progesterone,
testosterone, and DHT were significantly higher
than in females (Table 1). For both sexes, progesterone levels during January were not significantly different from December levels.
For testosterone, a statistically significant difference existed between serum levels in female
and male skates, with males being higher during
December and January. However, at times other
than these 2 months, the observed ranges overlapped. In females, the testosterone levels rose significantly from December to January (Table 1) and
were highest during February (Fig. 2C). In males,
testosterone levels increased from December to
January, with the trend continuing into February (Table 1). The levels in males remained significantly higher than those in females.
A similar male/female pattern was observed for
DHT, with serum levels in males again significantly higher than those in females. During January, values in both female and male skates
increased. In the males, the concentrations
reached 6005.4 ± 1166.7 pg/ml.
Effect of the presence or absence of male
skates on serum steroid hormone
concentrations in female skates
Serum steroid hormone concentrations in three
groups of adult female skates were compared during one reproductive season. All females were collected one month prior to the start of the study.
Group 1 females were maintained in indoor tanks
in the absence of males. Group 2 females were
maintained in indoor tanks in the presence of
males. Group 3 females were maintained outdoors
with males. Because of space limitations, it was
TABLE 1. Serum steroid hormone differences between male and female skates during December and January†
Hormone
December
17β-Estradiol
Testosterone
DHT
Progesterone
January
17β-Estradiol
Testosterone2
DHT
Progesterone
Male
(n = 20)
Female
(n = 20)
Difference between
male and female
76.0
35,390.0
3,716.7
150.0
±
±
±
±
44.9
7,971.2
281.7
7.4
670.3
2,096.5
90.0
14.7
±
±
±
±
106.8
510.2
90.0
7.41
F > M*
M > F*,**
M > F*
M > F*
22.8
42,706.0
6,005.4
150.0
±
±
±
±
7.8
16,787.0
1,166.7
7.4
2,480.0
14,000.0
550.5
42.7
±
±
±
±
106.8
6,010.3
400.0
47.41
F > M*
M > F*
M > F*
M>F
†Units = pg/ml.
*Significant difference. Statistical information: degrees of freedom = 24; P = 0.001–0.047; t = 4.5 to –5.0.
**Mann-Whitney rank sum test, P = 0.001.
1
Because the brief, well-circumscribed rises in progesterone occur after each egg-laying event, the monthly median data do not accurately
portray reproductive events. They are not really seasonal and are best described in individual animals.
2
Testosterone concentrations in males were highest in December through February, varying from 35,000 to 42,000 pg/ml. November and
April levels were 9,000–10,000 pg/ml.
REPRODUCTIVE HORMONE CYCLES IN RAJA EGLANTERIA
not possible to maintain females outside without
the presence of males. Photoperiods for all groups
were similar but not identical, since the indoor
environment was maintained at 12 hr light, 12
hr dark, and the outdoor environment averages
11 hr light, 13 hr dark during the months of January and February. The temperature was maintained at 20°C for the two indoor groups (groups
1 and 2), and fluctuations between 18 and 20°C
occurred for group 3. All populations began to lay
eggs, but eggs laid by the indoor animals without
males were infertile.
In all groups, January and February testosterone levels were high, and short-duration progesterone elevations were observed (Table 2).
However, no significant differences were observed
between groups.
Concentrations of two serum hormones, 17β-estradiol and DHT, demonstrated distinct differences between group 1 (indoors, with no males)
and group 2 (indoors, with males) as compared to
group 3 (outdoors, with males) (Table 2). Significantly higher concentrations of 17β-estradiol and
DHT were observed during January–February in
females maintained with males (both indoors and
outside) compared to the indoor group without
males (Table 2).
Serum hormone concentrations during
sequential movement of eggs within the
reproductive tract
Sixty observations of egg positions were obtained prior to blood sampling. The egg locations
were categorized into five groups as defined earlier. A number of significant differences was established in serum concentrations of steroid
hormones among these five groupings of egg positions (Fig. 3). When no egg capsules were detected,
17β-estradiol levels (stage 1) were significantly
581
lower than when eggs were found either in the
area of the nidamental gland (stage 2) or partially
down the uterus (stage 3) (Fig. 3A). Similarly, testosterone and DHT levels were significantly lower
in stage 1 than when eggs were detected in stages
2 and 3 or in the cloaca (stage 4) (Fig. 3B and D).
In contrast, progesterone concentrations were significantly higher at stage 5 (immediately after egg
laying and up to 48 hr post-egg laying) than at
all other stages (Fig. 3C).
Hormone patterns in serially sampled
female skates
Fifteen serially sampled female skates (each
sampled on multiple occasions) demonstrated consistent patterns of reproductive hormones during
the sampling periods from October to June, which
spanned both reproductive and nonreproductive
periods. Figure 4 depicts the mean serum 17βestradiol and testosterone (±SEM) values of these
fifteen serially sampled females. 17β-Estradiol
levels rose gradually in all of these females beginning in November/December, reaching highest levels in mid-February. Testosterone levels
also increased steadily until January, then again
rose until March. Testosterone concentrations remained high during the egg laying process. There
was a statistically significant difference in 17βestradiol levels during February and March compared to the nonreproductive months of June and
October and in testosterone levels during January, February, and March compared to May, June,
and October (Mann-Whitney rank sum, P =
0.001). The high levels of testosterone observed
during January and February in these serially
sampled females were consistent with the high
levels obtained in January–February from individually sampled females (Fig. 2B). In contrast,
elevations in progesterone were only randomly
TABLE 2. Serum steroid hormone concentrations (pg/ml) during January–February in three groups of female skates1
Group 1:
indoors (females only)
mean ± SE
Group 2:
indoors (with males)
mean ± SE
Group 3:
outdoors (with males)
mean ± SE
17β-Estradiol
1,441.8 ± 295.2
3,567.1 ± 71.9
4,023.1 ± 511.9
Testosterone
DHT
9,220.9 ± 63.9
362.3 ± 74.8
11,330.8 ± 79.9
662.3 ± 64.7
14,530.0 ± 101.9
761.0 ± 141.9
Hormone
Progesterone
1
67.0
(median)
92.0
(median)
Statistical criteria: Kruskal-Wallis one-way ANOVA on ranks and Dunn’s test, P < 0.05.
67.0
(median)
Significant difference
Yes: Groups 3 and 2
greater than group 1
Q = 6.7, 4.1
No
Yes: Groups 3 and 2
greater than group 1
Q = 3.6
No
582
L.E.L. RASMUSSEN ET AL.
Fig. 3. Serum steroid levels during five stages of egg laying in the female skate. Code for stages: 1 = pre-ovulatory,
no egg capsule present; 2 = egg capsule present in vicinity of
nidamental gland; 3 = encapsulated egg present partially
down uterine tract; 4 = encapsulated eggs present in cloaca;
5 = eggs laid within 0–48 hr. Statistical analyses using
Kruskal-Wallis one-way ANOVA on ranks: (A) 17β-estradiol.
Stage 1 significantly less than stages 2 (Q = 3.2) and 3 (Q =
3.6), and stages 2 and 3 significantly greater than stages 1,
4, and 5. (B) Testosterone. Stage 1 significantly less than
stages 2 (Q = 3.3), 3 (Q = 5.3), and 4 (Q = 5.6); in addition,
stages 3 and 4 greater than stages 1, 2 and 5. (C) Progesterone. Stage 5 significantly greater than all other stages: 1 (Q
= 4.2), 2 (Q = 3.5), 3 (Q = 2.9), and 4 (Q = 2.9). (D)
Dihydrotestosterone. Stage 1 significantly less than stages 2
(Q = 3.2), 3 (Q = 4.6), 4 (Q = 4.6), and 5 (Q = 3.1).
detected, primarily in January through April. Interestingly, there was a significant correlation
(Pearson product moment) P = 0.69 between 17βestradiol and testosterone concentrations for the
months studied.
haps be related to the continual ovulations that
occur throughout the egg laying process.
Other species of elasmobranchs also demonstrate increased 17β-estradiol during critical female reproductive events. For example, in the
Atlantic stingray, Dasyatis sabina, two distinct elevations of 17β-estradiol were observed in females,
one prior to ovulation and the other prior to initiation of histotroph production by the uterus
(Snelson et al., ’97). These results contrast with
17β-estradiol elevations observed during the preovulatory period in the placental bonnethead shark
(Manire et al., ’95) and the depression of 17β-estradiol in the aplacental spiny dogfish during pregnancy (Callard et al., ’89).
Especially elevated levels of 17β-estradiol are
consistent with pre-ovulatory folliculogenesis. Beginning with the early work on the aplacental viviparous ray, Torpedo marmorata, of Lupo di
Prisco et al. (’67) and continuing with other researchers (Fasano et al., ’92), it was demonstrated
that 17β-estradiol and androgen levels increased
DISCUSSION
This study provides a quantitative description
of circulating steroid hormone concentrations in
female clearnose skates throughout their annual
reproductive cycle with special attention to fluctuations during the active egg laying period. 17βEstradiol concentrations are detectable all year,
with circulating levels increasing during ovarian
growth and prior to ovulation and onset of observed breeding within the captive population
(September, October, and November). Levels are
especially high during the early period of egg laying (January and February). With the exception
of December, the extended period of elevated levels of 17β-estradiol during diverse reproductive
events may result from receptor stability or per-
REPRODUCTIVE HORMONE CYCLES IN RAJA EGLANTERIA
Fig. 4. Serum hormone concentrations in 15 serially
sampled female skates, from October through June. Upper
graph: 17β-estradiol; lower graph: testosterone. Standard errors are indicated by bars. *Indicates significant elevations
during several months in comparison to June and October
levels (P < 0.001, Mann-Whitney rank sum test).
as females reached the ultimate maturational
stage before ovulation. In the oviparous lesser
spotted dogfish, high levels of 17β-estradiol prior
to ovulation suggest a functional role in yolk production or oocyte maturation (Craik, ’79). In the
small spotted catshark, Scyliorhinus canicula,
Sumpter and Dodd (’79) clearly demonstrated a
ten-fold seasonal variation in 17β-estradiol and
testosterone, with hormonal levels rising with the
recrudescence of the ovary. The elegant work by
Koob et al. (’86) demonstrated a “correlation of
rising estradiol levels with follicle development
during recrudescence and the preovulatory elevation of estradiol during egg laying cycles in the
little skate, Raja erinacea.”
This study reveals an interesting pattern of serum progesterone elevation, clearly seen in the
data (Fig. 3C). An elevation in progesterone oc-
583
curred after each oviposition of a pair of eggs and
was detected in both individually and serially
sampled female skates. These progesterone elevations are observable; they occur just after ovipositioning and thus may be occurring prior to
ovulation. Progesterone quite likely could be the
maturation-inducing steroid of the clearnose
skate. Even the monthly data demonstrate that
the greatest frequency of progesterone elevations
occurs early in the egg laying period when the
maximum number of eggs are laid; as a few eggs
are laid in June, these elevations may be detectable in monthly sampling (Fig. 2D). Preovulatory
elevations of progesterone in the little skate have
been correlated with encapsulation of eggs prior
to ovulation (Koob et al., ’86).
The correlation of hormone levels with egg laying stages suggests that during the period after a
pair of eggs is laid, short-term elevations in
progesterone affect encapsulation or the timing
of the next ovulation. This may not be an absolute synchrony, but rather the continuation of a
sequence since intervals between laying of egg
pairs can be as short as 2 days, with a mean interval of 4.5 days.
In contrast, serum testosterone in female clearnose skates assessed on a monthly basis (Fig. 2B)
does not rise during ovarian recrudescence or prior
to initial ovulatory events. However, testosterone
concentrations increase dramatically with the onset of breeding activity and remain high during
egg laying. We observed that serum testosterone
and DHT concentrations were only significantly
elevated during January and February, the period
of maximum copulations and initiation of egg laying. In these months, there was a synchrony of
17β-estradiol and testosterone elevations, with testosterone elevations often slightly preceding 17βestradiol increases. Testosterone elevations have
been observed during mating and gestation in
lemon sharks (Rasmussen and Murru, ’92; Rasmussen and Gruber, ’93), and, interestingly, in reproducing female Atlantic stingrays, testosterone
elevations precede 17β-estradiol elevations
(Snelson et al., ’97). The elevation in testosterone
prior to 17β-estradiol elevation revealed in the individual skates (Fig. 4) are not apparent in the
egg laying data; rather, 17β-estradiol elevations
slightly precede testosterone elevation during the
egg laying process (Fig. 3). Koob et al. (’86) and
Tsang and Callard (’87) demonstrated in vivo in
R. erinacea that elevated serum titers of 17β-estradiol and testosterone were characteristic of the
follicular phase, and by in vitro studies they
584
L.E.L. RASMUSSEN ET AL.
showed that testosterone is the predominant steroid product of the theca.
In their 1986 in vivo study of R. erinacea, Koob
et al. demonstrated that the average daily titer
of testosterone during egg production was less
than 4 ng/ml, whereas in the current study of R.
eglanteria, testosterone concentrations were of this
low order of magnitude only during stage 1 (preovulatory, no egg capsule present, but no recent
oviposition). During the subsequent egg laying
processes (stages 2–5, from capsule formation in
the nidamental gland to ovopositing), testosterone levels were greater than 10 ng/ml (Fig. 3).
Serum testosterone concentrations were significantly higher when encapsulated eggs were in the
uterus (stage 3) and in the cloaca (stage 4), stages
equivalent to the “egg retention” period reported
by Koob et al. (’86). Thus, these results in R.
eglanteria contrast with the findings in R. erinacea
(Koob et al., ’86) that testosterone levels reached
their lowest values at the time of capsule formation. Instead, sustained testosterone elevations in
the clearnose skate during egg laying stages suggest that this hormone has a functional role in
egg laying.
The comparison in single-time sampled females
and serially sampled individual females for serum
testosterone and 17β-estradiol during a ninemonth period demonstrated considerable similarity. Females from both data sets demonstrated
significantly higher testosterone from January
through April. 17β-Estradiol levels were significantly higher during January and February in
both groups. The patterns of elevations and declines for testosterone and 17β-estradiol were similar when data from the serially sampled animals
were compared (Fig. 4).
Comparison of hormonal data between female
skates, with and without the presence of males,
allows for the separation of one variable affecting
the reproductive process of egg laying, specifically
the laying of fertilized versus nonfertilized eggs.
Female skates maintained indoors without males
and under conditions of constant temperature and
photoperiod nevertheless demonstrated high serum progesterone during egg laying. Egg laying
continued although the eggs were infertile, suggesting that ovulatory events, egg production, and
oviposition are at least somewhat affected by
progesterone concentrations. Testosterone was
similar in both groups, suggesting testosterone
may also have a role in the egg laying process.
17β-Estradiol, however, was elevated in both the
indoor and outdoor animals in the presence of
males, but was 2–3-fold lowered in females without males, implicating its role in the events associated with mating and fertilization.
ACKNOWLEDGMENTS
Sincere appreciation is expressed to C.J. Walsh,
P.C. Blum, C. Harris, J. Kaiser, and C. Manire
for their assistance at the Mote Marine Laboratory. This research was supported by Biosphere
Research Corporation (L.E.L.R. and D.L.H.), the
Mote Marine Laboratory (C.A.L.), and The Disney
Wildlife Conservation Fund (C.A.L.).
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