Fundulus heteroclitus gonadotropin(s)

GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

Fundulus I. Homologous

Yu-WAI *Whitney Laboratory, University, Appleton,

Bioassay

PETER

LIN,*

67, 126- 141 (1987)

heteroclitus

Gonadotropin(s)

Using Oocyte Maturation and Steroid Isolated Ovarian Follicles MICHAEL

J. LAMARCA,t

AND ROBIN

Production

by

A. WALLACES*

University of Florida, St. Augustine, Florida 32086. fDepartment of Biology, Lawrence Wisconsin 54911, and $Department of Anatomy and Cell Biology, College of Medicine, University of Florida, Gainesville, Florida 32610 Accepted March 17, 1987

Isolated ovarian follicles from several species were cultured to develop an in vitro bioassay system for Fundulus heteroclitus gonadotropin. An extract of F. heteroclitus pituitaries, when tested in heterologous systems using follicles from Rana pipiens, Xenopus laevis, and Carassius auratus, was ineffective in provoking either germinal vesicle breakdown or steroid production. In a homologous system using F. heteroclitus follicles, F. heteroclitus pituitary extract was capable of inducing both germinal vesicle breakdown and steroid production in a dose-dependent fashion. Testosterone, estradiol-17P, and 17a-hydroxy,20P-dihydroprogesterone were detected in both the culture media and the follicle extracts after F. heteroclitus pituitary extract stimulation. The steroidogenic responses resulting from the pituitary extract stimulation were dependent on the size and stage of follicular development. Only large vitellogenic follicles (1.2- 1.4 mm diameter) were able to produce 17u-hydroxy,20P-dihydroprogesterone and testosterone. Small vitellogenic follicles (Cl.2 mm) were unresponsive to stimulation by F. heteroclitus pituitary extracts as scored by either germinal vesicle breakdown or production of 17cy-hydroxy,ZOP-dihydroprogesterone and testosterone. However, estradiol-17e production was detected in follicles of a much wider size range: Follicles as small as 0.8 mm diameter were responsive to F. heteroclitus pituitary extract stimulation and produced a large quantity of estradiol-17P. There was a marked seasonal sensitivity of F. heteroclitus follicles to pituitary extract stimulation in vitro. Follicles obtained from fish outside of the breeding season (January) were less responsive to stimulation by pituitary extract or steroid. The same preparation of pituitary extract was capable of provoking germinal vesicle breakdown in follicles obtained in May. Pituitary extracts prepared during October through January were also less potent than those prepared during the breeding season (Februry through September). We conclude that F. heteroclitus gonadotropin(s) shows a noticeable species specificity and that F. heteroclitus follicles exhibit both a season- and a size-dependent responsiveness to gonadotropin(s). Hence, with a judicious use of the appropriate types of F. heteroclitus ovarian follicles, we have been able to demonstrate that in vitro oocyte maturation and steroid production are sensitive, homologous bioassays for F. heteroclitus gonadotropin(s). Q 1987 Academic Press. Inc.

One of the main obstacles to the isolation of fish gonadotropin (GtH) is the lack of a reliable and sensitive bioassay method to monitor biological activities during the purification procedure. A variety of biological techniques have been developed to check for gonadotropic activities. Tilapia GtH (Farmer and Papkoff, 1977) and pike eel GtH (Huang et al., 1981), for example, have been assayed using cultured rat

Leydig cells. Apart from the mammalian bioassay, an avian bioassay has been used for fish GtH. This involved the use of l-day-old cockerel testicular radiophosphate uptake for the isolation of salmon GtH (Donaldson et al., 1972; Idler et al., 1975b; Pierce et al., 1976; Huang et al., 1981). Amphibian bioassays using spermiation and oocyte maturation have been used to monitor gonadotropic activities of carp 126

0016-6480187

$1.50

Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

HOMOLOGOUS

BIOASSAY

and sturgeon pituitaries during the isolation procedure (Burzawa-GCrard, 1971; Burzawa-GCrard et al., 1975). A variety of piscine assays have also been developed. Goldfish gonads underwent hydration in response to carp pituitary extract injection (Clemens and Grant, 1964). Spermiation of goldfish was used as a bioassay for salmon GtH (Yamazaki and Donaldson, 1968). In vitro medaka oocyte maturation has been employed as a bioassay for a number of teleost GtHs (Hirose, 1980). The stimulation of gonadal cyclic adenosine-3’:5’-monophosphate in immature trout was used by Idler et al. (1975a) as a bioassay for chum salmon GtH isolation. Murrel ovarian mitochondrial cholesterol depletion was also employed as a bioassay for salmon, tilapia, and sturgeon GtH (Santanu et al., 1985). Recently, Kobayashi et al. (1985), using a rose bitterling ovipositor bioassay in conjunction with a radioreceptor assay, have succeeded in the purification of silver carp GtH. All of the above-mentioned bioassays involved the use of recipient species that were different from those of the pituitary donors. With these type of heterologous systems, it is not surprising to observe wide discrepancies in the potency of fish GtHs when tested on different bioassay systems (Fontaine et al., 1972; Goetz and Bergman, 1978; Epier et al., 1979; Idler and Ng, 1979). These discrepancies can be explained in part by the species specificity of the fish GtH. To circumvent the problem of species specificity, it is therefore expedient to use the same species (homologous system) for both the donor and the recipient of the GtH and to avoid, as much as possible, across-species testing (heterologous system). Such a homologous bioassay system ‘has been developed recently for the purification of coho salmon gonadotropin (Swanson et al., 1987). The ultimate objective of the present investigation was to develop an in vitro, homologous bioassay system for the isolation

FOR Funduhs

GONADOTROPIN

127

of Fundulus heteroclitus GtH(s). Numerous natural circumstances by happenstance have made F. heteroclitus an excellent candidate for such an endeavor. In particular, basic aspects of female reproduction and the mechanisms of oocyte growth, vitellogenesis, oocyte maturation, and oogenesis in general have been well documented for F. heteroclitus (Wallace and Selman, 1978, 1980, 1985; Selman and Wallace, 1983, 1986; Wallace and Begovac, 1985; Greeley et al., 1986; Selman et al., 1986). Based on this previous knowledge, we proceed to optimize an ovarian follicle culture and use it as a bioassay for F. heteroclitus gonadotropic activities. MATERIALS

AND METHODS

Kill&h (F. heteroclirus) were collected in minnow traps from saltmarshes in the Matanzas River-Intracoastal Waterway near the vicinity of the Whitney Laboratory (St. Augustine, FL). Captured fish were held in indoor fiberglass tanks with running seawater and were fed at least twice a day with dry flake food (Nutra Fin, R. C. Hagen Corp.) Occasionally, the diet was supplemented with mullet roe and shrimp. For heterologous bioassays, gravid female leopard frogs (Rana pipiens), South African clawed toads (Xenopus laevis), and goldfish (Carassius auratus) were obtained from commercial suppliers and maintained for a short time in the laboratory before use. R. pipiens were kept at 4” in a state of artificial hibemation (Lin and Schuetz, 1983). X. laevis were maintained in running well water at room temperature. C. awatus were kept at 15” in freshwater aquaria. Hormones. Equine luteinizing hormone (eLH; lot lO4F-08571), human chorionic gonadotropin (hCG), 17a-hydroxy,20@-dihydroprogesterone (17a-OH,20@DHP), estradiol-17P (E), testosterone (T), and progesterone were obtained from a commercial source (Sigma). Ovine luteinizing hormone (oLH; NIADDKoLH-25; 2.3 U/mg; 1 U = 1 mg NIH-LH-SI) was obtained from the National Hormone and Pituitary Program (Baltimore, MD). eLH and oLH (in lyophilized form) were reconstituted in 75% Liebovitz L-15 culture medium (with glutamine, Sigma). 17a-OH,20@ DHP was dissolved in ethanol vehicle. Various doses of steroid were added directly into the culture medium in a constant volume of vehicle (10 pl ethanol). F. heteroclitus pituitary extract (FPE). The bulk of the work described in this paper was carried out using pituitaries from female killifish. For the sake of comparison, pituitaries from male killifish were also colAnimals.

128

LIN,

LAMARCA,

lected. Due to a report of sexual differences in GtH (Breton et al., 1978), pituitaries from both sexes were not mixed. Pituitaries were isolated with watchmaker’s forceps from freshly killed animals after exposing the brain by cutting away the roof of the mouth. Isolated whole pituitaries were used fresh or stored frozen at -20”. For the preparation of FPE, pools of pituitaries were homogenized in cold 75% L-15 medium. The homogenates were then centrifuged for 15 min at 13,OOOgat 4” and the supernatants were collected. The remaining pellets were extracted once more with 75% L-15 and recentrifuged. Supematants from both extractions were combined and adjusted with 75% L-15 medium to a final concentration of 10 pituitary equivalents (pit equiv) per ml (e.g., 200 whole F. heteroclitus pituitary glands homogenized in 20 ml of 75% L-15 medium give a final concentration of 10 pit equiv/ml). The adjusted FPE was then subdivided into small tubes (5 pit equiv per tube) and stored frozen at -80”. Aliquots of frozen FPE were thawed before each experiment and used within 1 day. During the peak of the breeding season (May to August), several large pools of FPE were prepared, standardized against eLH (Fig. 2), and stored frozen for later use. These frozen FPE standards were used in all experiments unless stated otherwise. Preliminary experiments showed that whole pituitaries and FPE stored at -20 and -8O”, respectively, retained their biological activities for at least 1% years. Ovarian follicle culture. F. heteroclitus females were killed by decapitation and ovaries were immediately removed and put into a culture medium. Initial experiments (Fig. 1) indicated that late vitellogenic follicles (1.3-1.4 mm diameter) underwent a greater maturational response to both FPE and 17a-OH,208DHP when incubated in 75% L-15 (Greeley et al., 1986) rather than in solution FO originally developed for F. heteroclitus follicles (Wallace and Selman, 1978), so that 75% L-15 medium, containing 100 pg gentamicin/ml and adjusted to pH 7.5 with HCI, subsequently was used for all operations and cultures. Intact follicles were isolated from ovaries with fine watchmaker’s forceps. Follicles generally were pooled from 5 to 32 animals, depending on the ovarian condition and experimental requirement. Follicle diameters were measured with a dissecting microscope equipped with an ocular micrometer. The measured follicles were distributed randomly into 24.well tissue culture trays (Costar 3525). Usually, each culture well contained about lo-20 follicles in 2 ml medium. Incubation was carried out at 20” for 48-96 hr in a temperature-controlled incubator. The occurrence of germinal vesicle breakdown (GVBD) was utilized as an indication of oocyte maturation. At various timepoints (6- to 12-hr intervals), GVBD was scored to obtain a time course of the progression of oocyte maturation. To monitor the steroidogenic abilities of follicles in culture, samples of medium and methanol (HPLC grade)

AND WALLACE FPE

I7n-OH,20P-DPH

IOO-

80.

E

60-

5 08

40.

20

0 l-h Medium:

FO L-15 0 (pft. equiv. /ml)

(pq/ml)

FIG. 1. Response of isolated follicles to FPE and 17a-OH,20B-DHP in solution FO and 75% L-15 medium. Follicles (1.3- 1.4 mm diameter) were removed from six ovaries in 75% L-15 medium, randomized, and divided in half, and then each half was placed either in solution FO or 75% L-15 medium. Each half was further divided into four treatment groups and, for each treatment, 10 follicles were placed in each of six wells containing 2 ml of the appropriate solution with the indicated hormones. After 72 hr, percentage GVBD was scored for each well. Data bars indicate the average * SE for six wells.

extracts of follicles were collected separately, at specitied times, and were stored at -20” for later analysis of steroid content (Snyder and Schuetz, 1973; Lin and Schuetz, 1985). Steroid radioimmunoassay (RZA). Antisera for E and T were obtained from a commercial source (Wien Laboratory, Inc). Antiserum against 17a-OH,20BDHP was generously provided by Dr. Y. Nagahama; cross reaction with 20B-hydroxyprogesterone was 0.028%, while those with 17a-hydroxyprogesterone, progesterone, T, and E all were
HOMOLOGOUS

BIOASSAY

thin-layer chromatography (benzene:acetone, 4: 1). With this particular lot of enzyme, nearly complete conversion (>95%) of [3H]17~-hydroxyproge~terone to [3H]17a-OH,20P-DHP was obtained within 1 hr. General RIA procedures were modified from those of Lin and Schuetz (1985). Routinely, two sets of steroid standards were included in each assay. Logit-log transformation (Rodbti and Lewald, 1970) was used to linearize the standard curve. Logit transformation, regression line calculation, and sample logit conversion into picograms steroid were carried out with a personal computer.

RESULTS Heterologous bioassay of F. heteroclitus GtH(s). Preliminary experiments were carried out initially to search for a recipient species that is responsive to F. heteroclitus GtH(s). Several common standard laboratory species (R. pipiens, X. faevis, C. auratus) were utilized. Isolated ovarian follicles were cultured and monitored for their response to F. heteroclitus FPE. Results of these experiments are summarized in Table 1. eLH, progesterone, and 17~x-OH,20PDHP all were able to stimulate oocyte maturation in follicles from all species tested. FPE was unable, however, to promote either GVBD or progesterone production (data not shown) in heterologous follicles. In some experiments, the concentration of FPE was increased to as high as 7.5 pit equiv/ml to no avail. Among the various

BIOASSAY

OFF.

129

FOR Funduius GONADOTROPIN

species used, only F. heteroclitus follicles responded to FPE (Table 1). F. heteroclitus ovarian follicle dose response to GTHs: Comparison between FPE and eLH. The responses of F. heteroclitus ovarian follicles to homologous FPE versus heterologous eLH were compared. Dose-response curves toward FPE and eLH were constructed using isolated F. heteroclitus ovarian follicles (Fig. 2). Both FPE and eLH, as well as oLH (data not shown), induced F. heteroclitus ovarian follicles to undergo GVBD in vitro in a dose-dependent fashion. From this comparison, it can be shown that 1 pit equiv of FPE is approximately equivalent to 1000 IU of eLH. We were also able to establish that 1 IU eLH is approximately equivalent to 4 x 10e3 pg NIADDK-oLH-25 (data not shown). Male versus female FPE. Male and female FPEs were compared using maturational responses from the same batch of isolated ovarian follicles, The results (Fig. 3) were similar to the previous dose-response data and indicated that both male and female FPEs were maximally active at or above a concentration of 0.25 pit equiv/ ml, while a 50-fold dilution to 0.005 pit equiv/ml rendered the FPEs inactive. Graded responses between these extremes

TABLE 1 heteroclitus GtH USING OOCYTE

MATURATION

in Vitro

Hormone Follicle source

FPE

eLH

Progesterone

17a-OH,20P-DHP

Rana pipiens Xenopus laevis Carassius auratus Fund&us heteroclitus

+

+ + + +

+++ +++ + ++

++ ++ ++ +++

Note. Appropriate-size follicles from each species were isolated (R. pipiens: 1.5 mm; X. laevis: 1.3 mm; C. 1 .O mm; F. heteroclitus: I .4 mm). Ovarian follicles were randomized and distributed into each culture well containing 2 ml 75% L-15 medium (20 follicles per well). Various doses of different hormones were tested (FPE: 0.05-7.5 pit equiv/ml; eLH: 5-500 IU/ml; progesterone: 0.015-1.5 p&nl; 17a-OH,20@-DHP: 0.015-1.5 pg/ml). GVBD was scored up to 96 hr after the addition of hormones. The ” +” and “-” signs indicate the degree of GVBD achieved by the highest hormone doses in three trials (-: 0%; +: 25-50%; + + : 50-75%; + + + : 75-100%).

auratus:

130

LIN, LAMARCA,

AND WALLACE 100,

701 60-

6050g 2 8

i

Tl

0.25

05

60. mo 2 8 40

40-

i

30-

1

20-

ow 0

0.005 5

[FPEI

. I 1 0.025 25

(pit. equiv./mt)

0.05 50

or [eLH]

0.25 250

t

0.5 500

Control

0.005

Fundulus FPE eLH

(IU/~I)

FIG. 2. Response of isolated follicles to various concentrations of FPE or eLH. Ovarian follicles (1.3- 1.4 mm diameter) were isolated in May and June from a total of 20 fish. After isolation, lo-20 random follicles were distributed into wells containing 2 ml 75% L-15 medium and the indicated levels of FPE or eLH. After 72 hr, percentage GVBD was scored for each well. Data points indicate the average f SE for five wells. For standardization, the same procedures indicated that 50% GVBD was produced by 0.37 &ml NIAADK-oLH-25 (data not shown).

suggested that at lower doses female FPE may be more potent, but that at higher doses both male and female FPEs have the same activity. Seasonal variation in the gonadotropic activities of the female pituitary. The go-

nadotropin activities of each monthly pool of FPE collected throughout the year were tested with the same batch of F. heteroclitus ovarian follicles in vitro. Results of these experiments are depicted in Fig. 4. The ability of the various monthly pools of pituitary to induce GVBD followed closely the local F. heteroclitus reproductive season, which lasts from approximately February through September (Greeley et al., 1986). A gradual drop in gonadotropic activity was observed starting after September, and the lowest activity was reached in December. An increase in the biological activity during January brought

0.025

Pituitary

0.05 Extract

(pft. equiv /ml)

FIG. 3. Potency of FPE in stimulating oocyte maturation in vitro: Male versus female pituitary. Male and female pituitaries were collected separately from April through June. The whole pituitaries were stored frozen at -20” and extracts were prepared right before the beginning of the experiments in June. Follicles (1.2- 1.4 mm diameter) were isolated from a total of 15 fish and lo-15 follicles were distributed randomly into each culture well containing 2 ml of 75% L-15 medium together with various doses of male and female FPE. Oocyte maturation (percentage GVBD) was evaluated 72 hr later. Data bars indicate the average percentage GVBD 2 SE for four wells.

the potency back to maximum levels from February through September. It is interesting to note that with the higher concentration of FPE used (0.25 pit equiv/ml), the pituitaries collected in December were still capable of inducing more that 50% GVBD in vitro. Comparison of FPE and ovarian follicles collected at different times of the year: Time course and dose response. To have a

better understanding of the seasonal variation in gonadotropic activities, ovarian follicles and pituitaries from F. heteroclitus collected in January were compared with those collected in May. Time-course studies of in vitro oocyte maturation were carried out and the results are depicted in Fig. 5. At a low FPE concentration (0.05 pit equiv/ml), FPE obtained in January was not active whereas FPE obtained in May was capable of inducing about 50% GVBD

HOMOLOGOUS

P 2 0s

BIOASSAY

FOR Fundulus

131

GONADOTROPIN

60(pit. equiv. /ml)

e--e -

40-

0.250 0.025

20-

o-”

Control I Jun

Jul

Aug

Sep Month

Ott

Nov

Pituitories

Dee

Jan

Feb

Mar

Apr

1 May

Collected

FIG. 4. Seasonal variation in the gonadotropic activity of the pituitary. Female pituitaries were collected from June 1985 to May 1986. Pituitaries obtained during each month were pooled separately and stored frozen (- 20”) until the start of the culture experiments in May 1986. Ovarian follicles (1.3-1.5 mm diameter) were isolated from a total of 14 fish and lo-15 follicles were distributed randomly into separate culture wells containing 2 ml 75% L-15 medium together with two different doses (0.025 and 0.25 pit equiv/ml) of FPEs freshly prepared from the monthly pituitary collections. Oocyte maturation (percentage GVBD) was evaluated for each well 70 hr later. Each point represents the average percentage GVBD f SE for four culture wells.

in the same pool of ovarian follicles after 70 to 90 hr of culture (Fig. 5A). However, the time course of GVBD elicited by a moderate dose of steroid (17~OH,20B-DHP) was much faster, and the steroid was more effective in provoking GVBD when compared with the induction by a low concentration of FPE (Fig. 5A). Interestingly, the differences between the two pituitary preparations became less distinct when a higher dose of FPE (0.5 pit equiv/ml) was used. In this case, FPE obtained in January was as potent as that obtained in May (Fig. 5B). Moreover, the time courses of both the pituitary preparations were shifted to the left and their maturation-initiating activities approached the high efficacy displayed by the steroid. The dose response of ovarian follicles to different pituitary extracts is shown in Fig. 6. January follicles were essentially unresponsive to all doses of January FPE (Fig. 6A), but May follicles responded well to a high dose (0.5 pit equivlml) of January FPE

(Fig. 6B). A lower dose (0.05 pit equiv/ml) of January FPE was inactive on May follicles (Fig. 6B), but the same follicles gave better than a 50% response to the same dose of May FPE (Fig. 6C). For comparative purposes, follicles obtained in both January and May were also treated with a moderate dose of 17c+OH,20B-DHP, and May follicles were found to respond about twice as well as those obtained in January (Figs. 6D and E). Taken together, these results confirm that there is a seasonal fluctuation in gonadotropic potency of the pituitary, and further indicate that there is a seasonal difference in the responsiveness of follicles to both GtH and steroid. Time course of steroid production by F. heteroclitus ovarian follicles in vitro. The time course of steroid production by ovarian follicles (1.3 - 1.4 mm diameter) in response to FPE stimulation (0.25 pit equiv/ml) was monitored by RIA. At each specified time, culture media and follicle extracts were collected separately and

132

LIN, LAMARCA,

AND WALLACE

A

100 1

FPE

FPE

0.05

0.5

TT

pit. equiv./ml

pit. equiv.

1

/ml

Time

(hr)

5. Time course of oocyte maturation: Comparison of pituitaries collected in January and May at low and high doses. Female pituitaries collected in January and May were stored frozen at -20” until the experiments were carried out in May, at which time fresh extracts (FPE, and FPE,, respectively) were made. Ovarian follicles (1.2- 1.4 mm diameter) were isolated from a total of eight fish and 20 random follicles were placed in individual wells containing 2 ml 75% L-15 medium together with either 17a-OH,20g-DHP (0.015 &ml) or FPE at a concentration of 0.05 pit equiv/ml (A) or 0.5 pit equiv/ml (B). Oocyte maturation was scored for a period of 96 hr. Each point represents the average percentage GVBD + SE observed for four wells. FIG.

HOMOLOGOUS Hormone: Follicle

FPEJ I

FOR

Fundulus

133

GONADOTROPIN

FPE,

I7a-OH,20/3-DHP


: Jan

I00

s g

BIOASSAY

MOY

Jon

May

A

50

z

0

(pit.

equiv

/ml1

(pit. equiv. /ml)

(bq/ml)

FIG. 6. Response of follicles collected in January or May to FPE and 17~OH,20S-DHP. Extracts were prepared from pituitaries collected in January (FPE,) and May (FPE,) and immediately frozen at - 80”. Follicles (1.3- 1.4 mm diameter) were isolated from ovaries in both January (four fish) and May (five fish), 20 random follicles were incubated in wells containing 2 ml 75% L-15 medium together with various concentrations of the indicated hormones, and the percentage GVBD in each well was monitored 72 hr later. Each bar represents the average percentage GVBD + SE for three wells. The responses of January (A) and May (B) follicles to FPE,, May follicles to FPE, (C), and January (D) and May (E) follicles to 17~OH,208-DHP are shown.

stored frozen for later steroid analyses. Results are depicted in Figs. 7A-C. For all three steroids measured (T, E, I~cYOH,20@DHP), hormone levels eventually were higher in the culture media than those in the follicle extracts. Peak steroid accumulation occurred at around 30 to 40 hr after FPE addition. In control groups (without FPE addition), no measurable levels of T and 17r.x-OH,20@DHP were detected, while low background levels of E were detected in both the media and the extracts (data not shown). When oocyte maturation was monitored for this group of follicles, the time course of GVBD was found to lag behind maximum steroid accumulation by about 20 hr (Fig. 7D). Based on these data, steroid levels in culture media were measured routinely 36 hr after FPE addition in subsequent experiments. Steroid production by F. heteroclitus ovarian follicles in vitro: Relationship of follicle size to concentration of FPE. Isolated ovarian follicles of different sizes were cultured separately and the steroido-

genie responses of the various-size follicles to FPE stimulation were monitored. Steroid (T, E, 17a-OH,20@DHP) concentrations in the media, after 36 hr of culture, were measured by RIA. Results of the dose responses by various-size follicles to FPE stimulation are depicted in Fig. 8. The striking observation from this set of experiments was that the steroidogenic responses to pituitary stimulation were very dependent on the size and developmental stage of the ovarian follicle. Vitellogenic follicles of less than 1.1 mm diameter produced very little T (Figs. 8D and E, column I) or 17oOH,20@DHP (Figs. 8D and E, column III>. These same vitellogenic follicles were, however, responsive to FPE stimulation in terms of E production (Figs. 8D and E, column II). The unstimulated baseline levels of E were high in all stages of follicular development. In the cases of T and 17a-OH,20@DHP, no detectable levels of steroid were measured in unstimulated controls (zero FPE), regardless of follicle size. In the largest vitellogenic follicles

134

LIN, LAMARCA, IOOO-

A

800Testosterone

600: .+

400200-

r" &

8000-

2

7000.

; .g 5 LL .c c 1 0 f :: t a

6000. 5000. 4000. 30002000I ooo-

,ooo-

I7a-OH,20P-OHP

“‘1

D

8 2 c 0

6

12

18

24

30

Time

(hr)

36

42

48

72

7. Time course of steroid production by ovarian follicles in vitro. Ovarian follicles (1.3-1.4 mm diameter) were isolated in June from 17 fish and 20 random follicles were placed in each well containing 2 ml 75% L-15 medium together with FPE (0.25 pit equiv/ml). At each timepoint, oocytes were scored for percentage GVBD, and medium samples and methanol extracts of follicles were prepared separately and stored at -20” for later steroid RIA analyses. Indicated are the results for T (A), E (B), and 17a-OH,20B-DHP (C) determinations together with percentage GVBD (D). Each point represents the average + SE found for three,wells. FIG.

AND WALLACE

(1.2- 1.4 mm diameter), dose-response relationships to FPE concentration were established for all three steroids measured down to an FPE concentration as low as 0.005 pit equiv/ml (Figs. 8A and B, columns I-III). Among the steroids measured, E was the most abundant: E concentrations were about 10 times higher than T or 17a-OH,20@DHP levels (Fig. 8, column II). The results depicted in Fig. 8 were obtained with follicles isolated from ovaries that did not contain large preovulatory follicles (1.4-1.7 mm diameter). In another set of experiments, follicles were collected from fish that had preovulatory follicles in their ovaries. The steroidogenic responses of the preovulatory follicles were then compared with the smaller sister follicles isolated from the same pool of ovaries. The results are presented in Fig. 9. The preovulatory follicles (1.4- 1.7 mm) underwent spontaneous maturation (GVBD) in most instances by 36 hr of culture without exogenous stimulation. Interestingly, the doseresponse relationships to FPE stimulation were not well defined in these follicles in terms of T and E production (Figs. 9A and B, columns I and II). The smaller preovulatory follicles generated 17~OH,20@-DHP in response to FPE (Fig. 9B, column III), but the larger preovulatory follicles did not (Fig. 9A, column III). Vitellogenic follicles (1.1-1.4 mm diameter) were similar to those in the previous experiment in terms of T and 17~OH,20B-DHP production, but with synthesis being somewhat less (Figs. 9C and D, columns I and III). Again, however, no clear dose-response relationship was seen for E production by vitellogenic follicles and background levels were particularly high (Figs. 9C and D, column II). DISCUSSION

Results from this report demonstrate that a direct and sensitive in vitro bioassay for teleost GtH, using homologous ovarian fol-

I.

Testosterone

II.

10001

Estrodiol

- 17/3

III.

I7a-OH,20fi-DHP

8001

1

6000 800 400

600 400

4000

A. 1.3-1.4mm

600

600

400

6000 4000

200

2000

400

4000

200

2000

400

4000

200

2000

200

2000

1 8. 1.2-l.3mm

200

--

f\ E n 0 ‘0 5 E s 8 .; is 07

ND

L400

ND

D. I.O-l.lmm

E. 0.8-LOmm

ND ND

j NCI N,D N,D N,D

4000

1

C. I.1 -1.2 mm

200

1 F. Media

2ooj~,D

N,D N,D N,0

0 ,005 .05 .5 Fun&/us

““]N,D

~,0

up,

0 .005.05 Pituitary

Extract

j N.”

up

N,D N.D N,D

0 .005 .05 .5

.5 (pit.

Control

equiv./ml)

FIG. 8. Steroid production by ovarian follicles in vitro: Effects of follicle size and FPE dose on follicles isolated from ovaries without preovulatory follicles. During the latter part of the reproductive season (June to September), various-size ovarian follicles were isolated from 32 ovaries that did not have preovulatory (maturational) follicles (1.4- 1.7 mm diameter). Isolated follicles (0.8- 1.4 mm diameter) were measured and grouped according to size (A-E), and then 20 random follicles of each size were placed in culture wells containing 2 ml 75% L-15 medium together with the indicated concentrations of FPE. Media controls (F) consisted of 2 ml medium together with FPE but without follicles. After 36 hr, media from each well were collected, frozen, and later analyzed by RIA for T (column I), E (column II), and 17~OH,206-DHP (column III). Data bars indicate the average values + SE for six wells. ND, not detectable.

136

LIN, LAMARCA, I.

Testosterone

II.

AND WALLACE

Estrodiol -17p

III.

800

4000

400

400

2000

200

A. 1.5-1.7mm (loo% span. Mot.1 ND L&A

1

.

4009

I7a-OH.20/3-DliP

ND

1

= r

800

> ,o 0 2 9 E’

400

2000

8

800

4000

.x ? 5

400

2000

200

800

4000

400

400

2000

200

a 1.4 - 1.5 mm (50% Spot-l. Mat.)

200

C. l.2-l.4mm

D. Id-1.2mm

0

.005

.05

.5 Fundulus

0

.005

Pituitary

.05 Extract

.5 (pit.

L

NO 0

ND .005

.05

.5

eguiv. /ml)

FIG. 9. Steroid production by ovarian follicles in vitro: Effects of follicle size and FPE dose on follicles isolated from ovaries with preovulatory follicles. Various-size ovarian follicles (1 . l- 1.7 mm diameter) were isolated in August from five ovaries that contained preovulatory follicles (1.4- 1.7 mm diameter). The protocol used was the same as that indicated for Fig. 8. At the beginning of the experiment, all oocytes had intact germinal vesicles; by the end of the experiment, all of the larger preovulatory follicles (A) and 50% of the smaller preovulatory follicles (B) had undergone GVBD. Data indicate the average f SE for three wells.

licles, can be established for F. heteroc&us. This finding supplements the extensive body of information that has already accumulated over the years for this widely used fish (Huver, 1973; DiMichele et al., 1986). Although our primary purpose was to develop a biological assay for F. heteroclitus GtH, information gathered in this report may also serve to define some basic endocrine aspects of the ovary. In these experiments, we tried to ascertain the effects that the stage of follicle development and the season may have on the steroido-

genie responses of follicles toward various doses of FPE, using in vitro techniques. Since the F. heteroclitus ovary frequently contains follicles of all sizes during the reproductive season (Wallace and Selman, 1978), it is a particulary suitable model for this type of study. Our data indicate that F. heteroclitus FPE exhibits a rather stringent species specificity (Table 1). Heterologous test systems using ovarian follicles from R. pipiens, X. laevis, and C. aura&s all were unresponsive. However, until a larger

HOMOLOGOUS

BIOASSAY

number of other species have been tested with the purified hormone, strict species specificity cannot be established for FPE. Although FPE was strikingly ineffective in provoking GVBD in heterologous follicles, they all nevertheless responded, in various degrees, to a mammalian GtH (eLH). These results are consistent with the observations by Bona-Gall0 and Licht (198 1) and Licht (1983), whose extensive comparative studies have revealed a high degree of variability in the hormonal specificity of gonadotropin. The only successful bioassay for FPE was achieved by using homologous follicles. Large vitellogenic follicles (1.3- 1.4 mm diameter) isolated from the F. heteroclitus ovary have been found previously to respond to exogenous hormonal stimulation (Wallace and Selman, 1978, 1980; Greeley et al., 1986). Such follicles, with a clearly visible germinal vesicle and a cytoplasm with noncoalesced oil droplets, normally will remain at an arrested stage when cultured in vitro, even for an extended period of time, and will not mature unless exposed to maturation-inducing substances. This contrasts with the larger sister follicles (diameter 3 1.5 mm) present in the ovary. In these larger follicles, the peripherally attached oil droplets are more coalesced and the follicles appear distinctly more translucent when compared with smaller-size follicles (Wallace and Selman, 1978; Selman and Wallace, 1986). These larger, more translucent follicles inevitably undergo spontaneous maturation without exogenous stimulation (Wallace and Selman, 1978; Greeley et al., 1986). Hence they are committed to complete the maturational process and are not suitable for assessing gonadotropic activities in vitro. By using vitellogenic follicles of appropriate size (1.3-1.4 mm diameter), we found that FPE was capable of inducing oocyte maturation in vitro in a dose-dependent fashion (Figs. 1 and 2). We also found that one pit equiv of FPE equals 1000 IU eLH in terms of maturation-inducing

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Fun&us

GONADOTROPIN

137

ability (Fig. 2). The time course of GtH-induced GVBD, when compared with steroid (17a-OH,20@DHP) induction, generally showed a slower pace of progression (Fig. 5). This result was not surprising, since it is generally believed that the biological action of GtH is not targeted directly on the oocyte itself (Smith, 1975). Instead, it is mediated through the release of steroid from follicle cells and the steroid, presumably 17a-OH,20B-DHP (Nagahama and Adachi, 1985; Greeley et al., 1986), in turn initiates oocyte maturation. Several variables that may affect the performance of the GtH bioassay were investigated. We demonstrated that the gonadotropic potency of the pituitary showed a marked seasonal fluctuation that coincided with the natural breeding season of F. heteroclitus (Fig. 4). In December, when the ovaries were in their regressed stage, the potency of the pituitary extracts showed a corresponding decrease in their maturation-inducing ability. This conclusion was derived by testing the frozen pituitary obtained in December, together with pituitaries collected and frozen at other times of the year, for their abilities to induce oocyte maturation (GVBD) in vitro in a batch of follicles collected in the middle of the breeding season, Thus, the changing seasonal pattern of the gonadotropic contents in the pituitary was tested on a constant source of follicles. However, we could not discern whether there was a corresponding change in the secretion rate of GtH in vivo. Histological and ultrastructural studies on gonadotropic cells in the pituitary of other teleosts have suggested that quantitative changes in plasma and pituitary GtH, at various stages of the reproductive cycle, correspond with changes in the ultrastructure of the gonadotropic cells in the pituitary (Robertson and Wexler, 1962a,b; van Overbeeke and McBride, 1967; Peute et al., 1978). Generally, the “globular” stage of gonadotropic cells correlated with a high titer of GtH content, whereas low levels of

138

LIN,

LAMARCA,

GtH coincided with predominantly cisternal gonadotropic cells (Nagahama and Yamamoto, 1969; Peute et al., 1978). Probably both the globular and the secretory vesicles represent the storage organelles of the GtH principle, but do not necessarily reflect variations in GtH release. Our results on the seasonal fluctuation in GtH potency also correlated, in a general way, with an older cytochemical study of the F. heteroclitus pituitary in which presumed gonadotropes (aldehyde fuchsin-positive basophiles) were found to be much diminished in the winter (Sokol, 1961). However, the animals used for this study were collected at a more northern locality where the length of the breeding season is much reduced compared with that of southern populations (Wallace and Selman, 1981), so that the results of this older study cannot be strictly correlated with our own results. Another source of variability inherent in the homologous F. heteroclitus bioassay system is the change in the sensitivity of the ovarian follicle toward GtH. Even with the same type of follicles at a similar stage of development, there were intrinsic differences in the degree of responsiveness when follicles isolated from different fish were compared at the same time of year (data not shown). It is therefore imperative to have a well-randomized pool of follicles when comparing treatment effects in any experiments. In addition, there is a general seasonal variation in the ovarian condition. Early in the year (January and February), most of the ovaries of F. heteroclitus are still not fully developed. There are, however, some vitellogenic follicles and occasionally some ovulated eggs present in vivo. Large vitellogenic follicles (1.3- 1.4 mm diameter) isolated at this time were relatively insensitive to both steroid and gonadotropic stimulation in vitro (Fig. 6). This insensitivity could result from a variety of factors. As one possibility, the responsivity of freshly isolated ovarian follicles in vitro may be influenced by other

AND

WALLACE

synergistic hormones that may have acted directly or indirectly on the follicles in vivo. It has been shown in F. heteroclitus, as well as in a variety of other fish (pike, rainbow trout, goldfish), that a low dose of cortisol, when applied together with GtH or pituitary preparations, can greatly enhance the response of follicles in vitro (Jalabert, 1976; Wallace and Selman, 1980). However, attempts to enhance the in vitro responsiveness of the follicles collected early in the breeding season (January) by using a variety of hormones (cortisol, eLH, hCG) were altogether unsuccessful (data not shown). Apart from the in vitro oocyte maturation (GVBD) assay, the steroidogenic responses of F. heteroclitus follicles were also utilized to serve as a biological assay for GtH. Unlike the occurrence of GVBD, which is an all-or-none phenomenon, steroid production in response to gonadotropic stimulation can serve as a more quantitative and sensitive bioassay by exploiting the use of the high sensitivity inherent in steroid RIA. The steroidogenic responses of F. heteroclitus ovarian follicles were found to be very dependent on the stage of follicular development (Figs. 8 and 9). A good dose-response relationship could be established provided the follicles of appropriate size (1.2-1.4 mm diameter) were used (Figs. 8A and B). It is important to note that although high steroid levels could be detected in preovulatory follicles (al.5 mm diameter), the dose-response relationships were inevitably lost (Fig. 9A). Hence, the large, more translucent follicles (a 1.5 mm diameter) are not suitable to use in the bioassay. Also, E is the predominant steroid produced in response to pituitary stimulation, and E production is elicited from a wide range of follicle sizes (from 0% 1.4 mm diameter). Nevertheless, the use of E steroidogenesis to assess the GtH concentration may sometimes be restricted due to high background (unstimulated) levels.

HOMOLOGOUS

BIOASSAY

FOR Fundulus

The profile of steroid production exhibited by the various-size follicles is very intriguing. In particular, T and 17~OH,20PDHP production can be significantly induced only in large (1.2- 1.4 mm diameter) vitellogenic follicles (Figs. 8 and 9). Oocytes contained in this type of follicle are also the only ones that are consistently responsive to 17ol-OH,20P-DHP stimulation in vitro (Greeley et al., 1986). It has been demonstrated in many teleosts that 17~ OH,20@DHP is the most effective steroid in stimulating oocyte maturation (Jalabert, 1976; Goetz and Theofan, 1979; Duffey and Goetz, 1980; Nagahama et al., 1983; Greeley et al., 1986). It has also been shown for amphibian follicles that there is a shift from estrogenic to progestational steroid production when follicles progress from vitellogenesis to a prematurational stage (Fortune, 1983). In goldfish, estradiol-17l3 production was much reduced in the prematurational stage (tertiary yolk stage) follicles when compared with the vitellogenic follicles (Kagawa et al., 1984). Maximal production of testosterone was also observed in these tertiary yolk stage follicles upon hCG stimulation (Kagawa et al., 1984). Recently, it was demonstrated in coho salmon that the concentration of estradiol-17P was reduced while the production of testosterone and 17~0H,20@DHP increased with advancing oocyte development (Van Der Kraak and Donaldson, 1986). However, such a shift is not detected in F. heteroclitus: The level of E is consistently higher in all stages of follicles tested (Figs. 8 and 9). Thus, high levels of E coexist with high progestagen levels despite the fact that E is considered to be inhibitory to oocyte maturation (Lin and Schuetz, 1983, 1985). Hence, the role of various steroids in the initiation of oocyte maturation in F. heteroclitus remains to be resolved. Although pituitary GtH in teleosts, as in most other vertebrates, is considered to be the primary endocrine factor controlling

139

GONADOTROPIN

oocyte maturation, its mechanism of action is only partially known. It seems likely that a pituitary-ovarian relay mechanism exists for the stimulation of final oocyte maturation (Goetz, 1983). It has been hypothesized that in most fish species, the synthesis of a maturational steroid in the follicle is the obligatory intermediate step for gonadotropic action (Jalabert, 1976; Hirose, 1976). The only exception reported thus far is the Indian catfish, Heteropneustesfossilis. In this case, the corticosteroids produced by the interrenal tissue in response to GtH stimulation appear to be the intermediate step required to induce oocyte maturation (Sundararaj and Goswami, 1977). The detection of 17oOH,20P-DHP in the culture media after GtH stimulation is consistent with the notion that this steroid, or a steroid with similar structure, is the maturation-inducing substance in F. heteroclitus (Greeley et al., 1986). However, we cannot rule out the possible existence of a pituitary-interrenal-ovarian relay for the stimulation of final oocyte maturation. In summary, regardless of the true mechanism of GtH action, the use of both oocyte maturation in vitro and, in particular, the steroidogenic responses of ovarian follicles are here demonstrated to be sensitive homologous bioassays for F. heteroclitus GtH(s) uncomplicated by the problems of species specificity. ACKNOWLEDGMENT This work was supported DCB-8511260 to R.A.W.

by NSF

Grant

REFERENCES Bona-Gallo, A., and Licht, P. (1981). Gonadotropin specificity of in vitro testosterone secretion by fish testes. Gen. Comp. Endocrinol. 43, 461-478. Breton, B., Prunet, I!, and Reinaud, P. (1978). Sexual differences in salmon gonadotropin. Ann. Biol. Anim. Biochim. Biophys. l&159-765. Burzawa-Gerard, E. (1971). Purification dune hormone gonadotrope hypophysaire de Poisson teleosteen, la carpe (Cyprinus carpio L.). Biochimie 53, 545-5.52.

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AND WALLACE fects of steroids on germinal vesicle breakdown of intact follicles in vitro. Gen. Comp. Endocrinol. 62, 281-289. Hirose, K. (1976). Endocrine control of ovulation in medaka (Oryzias latipes) and ayu (Plecoglossus altivelis).

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Huver, C. W. (1973). “A bibliography of the Genus Fundulus.” Hall, Boston. Idler, D. R., Hwang, S. J., and Bazar, L. S. (1975a). Fish gonadotropin(s). I. Bioassay of salmon gonadotropin(s) in vitro with immature trout gonads. Endocrinol. Res. Commun. 2, 199-213. Idler, D. R., Bazar, L. S., and Hwang, S. J. (1975b). Fish gonadotropin(s). II. Isolation of gonadotropin(s) from chum salmon pituitary glands using affinity chromatography. Endocrinol. Res. Commun. 2, 215-235. Idler, D. R., and Ng, T. B. (1979). Studies on two types of gonadotropins from both salmon and carp pituitaries. Gen. Comp. Endocrinol. 38, 421-440. Jalabert, B. (1976). In vitro oocyte maturation and ovulation in rainbow trout (Salrno gairdneri), northern pike (Esox lucius) and goldfish (Carassius

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974-988. Kagawa, H., Young, G., and Nagahama Y. (1984). In vitro estradiol-17B and testosterone production by ovarian follicles of the goldfish, Carassius auratus. Gen. Camp. Endocrinol. 54, 139-143. Kobayashi, M., Aida, K., and Hanyu, I. (1985). Radioimmunoassay for silver carp gonadotropin. Bull. Japan. Sot. Sci. Fish. 51, 1085-1091. Licht, P. (1983). Evolutionary divergence in the structure and function of pituitary gonadotropins of tetrapod vertebrates. Amer. Zool. 23, 673-683. Lin, Y.-W. P.. and Schuetz, A. W. (1983). In vitro estrogen modulation of pituitary and progesteroneinduced oocyte maturation in Rana pipiens. J. Exp. Zool. 226, 281-291. Lin, Y.-W. P., and Schuetz, A. W. (1985). Intrafollicular action of estrogen in regulating pituitaryinduced ovarian progesterone synthesis and oocyte maturation in Rana pipiens: Temporal relationship and locus of action. Gen. Camp. Endocrinol. 58, 421-435. Nagahama, Y., and Adachi, S. (1985). Identification of

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maturation-inducing steroid in a teleost, the amago salmon (Oncorhynchus rhodurus). Dev. Biol. 109, 428-435. Nagahama, Y., Hirose, K., Young, G., Adachi, S., Suzuki, K., and Tamaoki, B. (1983). Relative in vitro effectiveness of 17a,20P-dihydroxy-4pregnen-3-one and other pregnene derivatives on germinal vesicle breakdown in oocytes of four species of teleost, ayu (Plecoglossus altivelis), amago salmon (Oncorhynchus rhodurus), rainbow trout (S&no gairdneri), and goldfish (Curussius aura&s). Gen. Comp. Endocrinol. 51, 15-23. Nagahama, Y., and Yamamoto, K. (1969). Basophils in the adenohypophysis of the goldfish (Curassius auratus). Guma Symp. Endocrinol. 6, 39-55. Peute, J., Goos, H. J. Th., de Buryn Marcelle, G. A., and van Oordt, P. G. W. J. (1978). Gonadotropic cells of the rainbow trout pituitary during the annual cycle: Ultrastructure and hormone content. Ann. Biol. Anim. Biochem. Biophys. 18,905-910. Pierce, J. G., Faith, M. R., and Donaldson, E. M. (1976). Antibodies to reduced S-carboxymethylated alpha subunit of bovine luteinizing hormone and their application to study of the purification of gonadotropin from salmon (Oncorhynchus tshawytscha) pituitary glands. Gen. Camp. Endocrinol. 30, 47-60. Robertson, 0. H., and Wexler, B. C. (1%2a). Histological changes in the pituitary gland of the rainbow trout (Salmo gairdneri) accompanying sexual maturation and spawning. J. Morphol. 110, 157-170. Robertson, 0. H., and Wexler, B. C. (1962b). Histological changes in the pituitary gland of the Pacific salmon (genus Oncorhynchus) accompanying sexual maturation and spawning. J. Morphol. 110, 171-185. Rodbard, P., and Lewald, J. E. (1970). Computer analysis of radioligand assay and radioimmunoassay data. Acta Endocrinol. 64 (Suppl. 147), 79-103. Santanu, D., Jamaluddin, M., Bhattacharya, S., Bhadra, R., and Datta, A. G. (1985). Bioassay of fish gonadotropin by ovarian mitochondrial cholesterol depletion. Gen. Comp. Endocrinol. 57, 491-497. Selman, K., and Wallace, R. A. (1983). Oogenesis in Fundulus heteroclitus. III. Vitellogenesis. J. Exp. Zool. 226, 441-457. Selman, K., and Wallace, R. A. (1986). Gametogenesis in Fundulus heteroclitus. Amer. Zool. 26, 173-192. Selman, K., Wallace, R. A., and Barr, V. (1986). Oogenesis in Fundulus heteroclitus. IV. Yolk-vesicle formation. J. Exp. Zool. 239, 277-288.

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Smith, L. D. (1975). Molecular events during oocyte maturation. In “The Biochemistry of Animal Development” (R. Weber, Ed.), Vol. 3, pp. l-16. Academic Press, New York. Snyder, B. W., and Schuetz, A. W. (1973). In vitro evidence of steroidogenesis in the amphibian (Rana pipiens) ovarian follicle and its relationship to meiotic maturation and ovulation. J. Exp. Zool. 183, 333-342. Sokol, H. W. (1961). Cytological changes in the teleost pituitary gland associated with the reproductive cycle. J. Morphof. 109, 219-235. Sundararaj, B. I., and Goswami, S. V. (1977). Hormonal regulation of in vivo and in vitro oocyte maturation in catfish, Heteropneustes fossilis (Bloch). Gen. Comp. Endocrinol. 32, 17-28. Swanson, P., Dickhoff, W. W., and Gorbman, A. (1987). Pituitary thyrotropin and gonadotropin of coho salmon (Oncorhynchus kisutch): Separation by chromatofocusing. Gen. Comp. Endocrinol. 65, 269-287. Van Der Kraak, G., and Donaldson E. M. (1986). Steroidogenic capacity of coho salmon ovarian follicles throughout the periovulatory period. Fish Physiol. Biochem. 1, 179-186. van Overbeeke, A. P., and McBride, J. R. (1967). The pituitary gland of the sockeye (Oncorhynchus nerka) during sexual maturation and spawning. J. Fish. Res. Board Canada 24, 1791-1810. Wallace, R. A., and Begovac, P. C. (1985). Phosvitins in Fundulus oocytes and eggs: Preliminary chromatographic and electrophoretic analyses together with biological considerations. J. Biol. Chem. 260, 11268- 11274. Wallace, R. A., and Selman, K. (1978). Oogenesis in Fund&s heteroclitus. I. Preliminary observations on oocyte maturation in vivo and in vitro. Dev. Biol. 62, 354-360. Wallace, R. A., and Selman, K. (1980). Oogenesis in Fundulus heteroclitus. II. The transition from vitellogenesis into maturation. Gen. Comp. Endocrinol. 42, 345-354. Wallace, R. A., and Selman, K. (1981). The reproductive activity of Fund&us heteroclitus females from Woods Hole, Massachusetts, as compared with more southern locations. Copeia, 1981, 212-215. Wallace, R. A., and Selman, K. (1985). Major protein changes during vitellogenesis and maturation of Fundulus oocytes. Dev. Biol. 110, 492-498. Yamazaki, F., and Donaldson, E. M. (1968). The spermiation of goldfish (Carassius auratus) as a bioassay for salmon (Oncorhynchus tschawytscha) gonadotropin. Gen. Comp. Endocrinol. 10, 383-391.