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BOR - Papers in Press, published online ahead of print October 14, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.003749
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BIOLOGY OF REPRODUCTION 68, 328–335 (2003)
DOI: 10.1095/biolreprod.102.003749
© 2003 by the Society for the Study of Reproduction, Inc.

Ovary

Localization and Secretion of Inhibins in the Equine Fetal Ovaries1

Yumiko Tanakaa,b, Hiroyuki Taniyamac, Nobuo Tsunodad, Chandana B. Herathb, Rie Nakaia,b, Hiromi Shinbob, Natsuko Nagamined, Yasuo Namboe, Shun-ichi Nagataf, Gen Watanabea,b, Nigel P. Groomeg, and Kazuyoshi Taya2,a,b

a Department of Basic Veterinary Science, The United Graduate School of Veterinary Science, Gifu University, Gifu 501-1193, Japan b Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan c Department of Veterinary Pathology, Rakuno Gakuen University, Hokkaido 069-8501, Japan d Shadai Corporation, Hokkaido 059-1432, Japan e Equine Research Institute, Japan Racing Association, Tochigi 320-0856, Japan f Laboratory of Racing Chemistry, Tochigi 320-0851, Japan g School of Biological and Molecular Sciences, Oxford Brookes University, Oxford OX3 0BP, United Kingdom


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To clarify the source of inhibins in equine female fetuses, concentrations of immunoreactive (ir-) inhibin, inhibin pro-{alpha}C, and inhibin A in both fetal and maternal circulation and in fetal ovaries were measured. In addition, the localization of inhibin {alpha} and inhibin/activin ßA, and ßB subunits and the expression of inhibin {alpha}A and inhibin/activin ßA subunit mRNA in fetal ovaries were investigated using immunohistochemistry and in situ hybridization. Concentrations of circulating ir-inhibin, inhibin pro-{alpha}C, and inhibin A were remarkably more elevated in the fetal than in the maternal circulation between Days 100 and 250 of gestation. Fetal ovaries contained large amounts of ir-inhibin, inhibin pro-{alpha}C, and inhibin A. In contrast, these inhibin forms were undetectable in both the maternal ovaries and placenta. The inhibin {alpha} and inhibin/activin ßA and ßB subunit proteins were localized to enlarged interstitial cells of the equine fetal ovary. Expression of inhibin {alpha} and inhibin/activin ßA subunit mRNAs were also observed in the interstitial cells. We conclude that the main source of large amounts of inhibins in fetal circulation is interstitial cells of fetal ovary and is not of maternal origin. Furthermore, these inhibins may play some important physiological roles in the development of gonads in the equine fetus.

inhibin, ovary, pregnancy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inhibins, which belong to the transforming growth factor ß (TGFß) superfamily [1–5], were initially discovered as gonadal peptides that preferentially inhibit FSH secretion from the pituitary gland [6]. Inhibin consists of an {alpha} subunit linked by a disulfide bridge to one of the two highly homologous ß subunits (ßA and ßB) to form inhibin A ({alpha} and ßA) or inhibin B ({alpha} and ßB). Inhibins are mainly secreted by granulosa cells of the ovary in adult cyclic females of various mammals, such as rats [7, 8], hamsters [9], sheep [10], pigs [11], cows [11], monkeys [12], and humans [7]. In primates, other sources of inhibins include the corpus luteum and placenta during pregnancy [13, 14]. Granulosa cells of various sized follicles and theca cells of large follicles are the primary sources of inhibin during the estrous cycle of the mare [15–17]. Inhibin production in fetal ovaries has been studied in sheep [18–20], cows [21], monkeys [22], and humans [22]. A previous study by our laboratory [23, 24] and a study by others [25] have documented inhibin secretion and the localization of inhibin {alpha} subunit mRNA, respectively, in the fetal ovaries. Our previous paper [23] demonstrated that high concentrations of immunoreactive (ir-) inhibin were present in homogenates of equine fetal ovaries at Day 190 of gestation. Furthermore, immunohistochemical staining revealed the presence of inhibin {alpha} subunits in the interstitial cell of equine fetal ovaries. However, despite the high concentrations of ir-inhibin in homogenate of equine fetal ovaries, they failed to exert any suppressive bioactivity on FSH secretion by rat pituitary cells cultured in vitro. Therefore, the objective of the present study is to clarify the secretion of inhibins in equine female fetuses throughout the second half of gestation using four techniques: radioimmunoassay (RIA) of ir-inhibin; ELISA of inhibin pro-{alpha}C and inhibin A; immunohistochemistry of inhibin {alpha} and ßA and ßB subunits; and in situ hybridization of inhibin {alpha} and ßA subunit mRNAs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Blood Samples

Fifteen female fetuses were recovered from 25 Thoroughbred and Anglo-Arab pregnant mares at Days 100–250 of gestation (Days 100–150, n = 5; Days 150–200, n = 8; Days 200–250, n = 6) (term = 340 days). Final mating day was designated as Day 0 of gestation. The pregnant mares were given an overdose of a mixture of barbiturate (Ravonal; Tanabe Pharmaceutical Co., Ltd., Osaka, Japan) and suxamethonium chloride (Succine; Yamanouchi Pharmaceutical Co., Ltd., Osaka, Japan) and then the fetuses were recovered. The maternal blood samples were collected by venipuncture from the jugular vein, and the fetal blood samples were collected by cardiac puncture immediately after euthanasia. All blood samples were collected into plastic tubes containing heparin (20 IU/ml blood) as anticoagulant to prevent clotting. The samples were stored in ice and centrifuged at 1700 x g for 15 min at 4°C immediately after completing the experiment. The resulting plasma was harvested and stored at -20°C until assayed for inhibins. All procedures were carried out in accordance with the guidelines established by the Rakuno Gakuen University for use of laboratory animals.

Fetal Ovarian Samples

Ovaries from dams and fetuses were removed and weighed. Ovaries from 15 female fetuses were used for measurement of ovarian concentrations and tissue localization of inhibin proteins and mRNA. To measure inhibin concentrations, a portion of one ovary (1.0 g) was homogenized in 0.01 M PBS (pH 7.4). The homogenized ovarian samples were centrifuged at 20 000 x g for 30 min at 4°C. The supernatant was removed and stored at -80°C until assayed for inhibins.

The other ovary was fixed in freshly prepared 4% (w/v) paraformaldehyde (PFA; Sigma Chemical Co., St. Louis, MO) in 0.01 M PBS and embedded in paraffin. For immunohistochemistry and in situ hybridization, 5-µm- and 8-µm-thick tissue sections were prepared, respectively. These sections were placed on glass slides coated with 3-aminopropyltriethoxysilane.

Radioimmunoassay

Concentrations of ir-inhibin in plasma and ovarian homogenates were measured by a double-antibody RIA, as described previously [26]. Purified bovine 32-kDa inhibin from bovine follicular fluid was used as the standard. The same material was labeled with 125I according to the chloramine-T method. The antiserum used in this assay was raised against bovine 32-kDa inhibin (TNDH-1) used in castrated male rabbits in our laboratory. The assay system does not distinguish dimeric inhibin from {alpha} subunit monomer and inhibin pro-{alpha}C. Thus, inhibin levels are referred to as ir-inhibin in the present study [27]. We have validated the assay system for equine plasma as well as ovarian homogenates, as described previously [23]. In the previous study, we reported the parallelism tested for ir-inhibin in equine follicular fluid, pregnant and cyclic mare plasma, and homogenates of placenta, fetal ovaries, newborn foal ovaries, and newborn foal testes. The results were expressed in terms of 32-kDa bovine inhibin. The sensitivity of the assay was 7.8 pg/tube. Intra- and interassay coefficients of variation were 8.0% and 16.2%, respectively.

ELISA

Concentrations of inhibin pro-{alpha}C in plasma and ovarian homogenates were measured using a two-site ELISA kit (Serotec, Oxford, UK) designed for measurement of human inhibin pro-{alpha}C. In the assay, two monoclonal antibodies directed against the pro and {alpha}C regions were used [28]. The amount of inhibin pro-{alpha}C was expressed in terms of the purified inhibin pro-{alpha}C from human follicular fluid. The sensitivity of the assay was 0.15 pg/tube. Intra- and interassay coefficients of variations were 3.3% and 12.6%, respectively.

Concentrations of inhibin A in plasma and ovarian homogenates were measured using a two-site ELISA kit. The preparation of a new monoclonal antibody to the {alpha} subunit of cow inhibin (PPG1/14/6), together with E4 monoclonal antibody to the inhibin/activin ßA subunit, has produced an ELISA able to measure inhibin A in sheep [29], cow [30], and male equine fetus [24] with a similar sensitivity to the human inhibin A ELISA. The amount of inhibin A was expressed in terms of the purified 32-kDa bovine inhibin A from bovine follicular fluid. The sensitivity of the assay was 0.78 pg/tube. Intra- and interassay coefficients of variation were 4.2% and 8.7%, respectively.

Serial dilutions of pooled equine plasma and ovarian homogenates were assayed to test for parallelism.

Immunohistochemistry

After deparaffinization with xylene, the tissue sections were subjected to antigen retrieval by autoclaving in 0.01 M sodium citrate buffer, pH 6.0, at 121°C for 15 min. Sections were then incubated in 3% H2O2 in methanol at room temperature for 15 min, followed by Block Ace (Dainippon Pharmaceutical Co., Ltd., Osaka, Japan) at 37°C for 1 h to quench nonspecific staining. Then they were incubated at 37°C for 16–18 h with polyclonal antibodies at a dilution of 1:1000–3000. Antibody against each inhibin subunit was anti-[Tyr30]-porcine inhibin {alpha} chain (1-30)-NH2 conjugated to rabbit serum albumin ([Tyr30]-porcine inhibin {alpha} chain (1-30)-NH2 was kindly provided by Dr. N. Ling, Neuroendocrine Inc., San Diego, CA); monoclonal antibodies raised against the synthetic peptides corresponded to amino acid sequence of 82–114 of human ßB subunit (E4) or ßA subunit (C5). After incubation with the antibody, sections were treated with 0.25% (v/v) biotinylated goat anti-mouse secondary antibody (Elite ABC kit; Vector Labs, Burlingame, CA) in Block Ace at 37°C for 1 h. These sections were subsequently incubated with 2% (v/v) avidin-biotin complex (Elite ABC kit) in casein-Tris-saline (CTS) at 37°C for 30 min. The reaction products were visualized by treating with 0.025% (w/v) 3.3'-diaminobenzidine tetrachloride (Sigma) in 100 mM Tris-buffered saline containing 0.01% H2O2 for 1-30 min [15].

Complementary RNA Probes

The 1286-base pair (bp) equine inhibin {alpha} subunit cDNA clone [31] and the 1479-bp equine inhibin/activin ßA subunit cDNA clone [32] were subcloned into a pBluescript KS (-) vector (Stratagene, La Jolla, CA). Template inhibin {alpha} subunit and inhibin/activin ßA subunit cDNAs represent 309-bp subclones corresponding to the 5' region from the first restriction site PstI and 325-bp subclones corresponding to the 5' region from the first restriction site HindIII, respectively. For in vitro transcription of antisense probes, the Bluescript KS (-) vectors containing equine inhibin {alpha} and inhibin/activin ßA subclones were linearized with EcoRI, and digoxygenin (DIG)-labeled probes were synthesized with T3 RNA polymerase for inhibin {alpha} or with T7 RNA polymerase for inhibin ßA in the presence of DIG RNA labeling mixture (Roche Diagnostics, Tokyo, Japan) according to the procedures recommended by Roche Diagnostics. For the synthesis of DIG-labeled sense RNA probes, the Bluescript KS (-) vectors were linearized with XbaI for inhibin {alpha} or with HindIII for inhibin ßA. T7 RNA polymerase was used for synthesis of inhibin {alpha} sense probes, and T3 RNA polymerase was used for inhibin/activin ßA sense probes.

In Situ Hybridization

The sections were deparaffinized and rehydrated in the same way as for immunohistochemistry and were washed in 0.1 M Tris-HCl (pH 7.5). The sections were treated with 0.3% Triton X-100 in 0.01 M PBS (pH 7.4) for 10 min, digested with 50 µg/ml proteinase K (DAKO Japan Co., Kyoto, Japan) for 20 min at 37°C, treated with 0.2% glycine in 0.1 M Tris-HCl, prefixed in 4% PFA in 0.01 M PBS, and treated with 0.2% glycine in 0.1 M Tris-HCl. The sections were washed in 0.1 M Tris between treatments. The sections were completely dried and prehybridized in hybridization buffer (50% deionized formamide, 20x saline sodium citrate [SSC], 1 M Hepes [pH 7.0], 50x Denhardt solution, 100 µg/ml salmon sperm DNA) for 30 min at 37°C. Complementary RNA probes diluted in hybridization buffer were denatured for 10 min at 70°C and hybridized for 18 h at 55°C under coverslips in a humidified box.

After hybridization, the sections were washed in 4x SSC for 3 min at room temperature, 4x SSC for 20 min at 42°C, 2x SSC for 30 min at 65°C (twice), and 0.1x SSC for 30 min at 70°C (twice). The sections were washed in buffer 1 (0.1 M Tris-HCl, 0.15 M NaCl, pH 7.5) for 5 min, incubated with Block Ace for 20 min at 37°C, followed by alkaline phosphatase-conjugated anti-DIG IgG (1:500, Roche Diagnostics) for 1 h at 37°C. After washing buffer 1 for 5 min and buffer 3 (0.1 M Tris-HCl, 0.1 M NaCl, 0.05 M MgCl2, pH 9.5) for 10 min at room temperature, cRNA probes were visualized using nitroblue tetrazolium salt (Roche Diagnostics) and 5-bromo-4-chloro-3-indolyl phosphate (Roche Diagnostics). The reaction was stopped in water, and the sections were mounted.

Statistical Analysis

One-way analysis of variance (ANOVA) was carried out, and the significance between means was determined by a Duncan multiple range test. Data are presented as means ± standard errors (±SEM). A probability value less than 0.05 was considered to be significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Weight of Maternal and Fetal Ovaries (Table 1)

At Days 100–150 of gestation, the mean weight (±SEM) of fetal ovaries was 26.2 ± 6.3 g (n = 5) and that of maternal ovaries was 61.1 ± 13.8 g (n = 5). At Day 150–200 of gestation, the mean weight of fetal ovaries was 89.6 ± 9.1 g (n = 8) and that of maternal ovaries was 48.7 ± 5.0 g (n = 8). At Days 200–250 of gestation, the mean weight of fetal ovaries was 114.7 ± 18.5 g (n = 6) and that of maternal ovaries was 43.0 ± 7.6 g (n = 6). The fetal ovaries were significantly heavier (P < 0.05) than maternal ovaries at Days 150–200 and 200–250 of gestation.


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  		TABLE 1. Weights of fetal and maternal ovaries during gestation in the horses.*

Characterization of the Inhibin Pro-{alpha}C and Inhibin A ELISA Systems

Dose-response curves of serially diluted plasma and ovarian homogenate obtained from fetuses at 200 days of gestation produced suppression curves that were parallel to the standard curves produced with human inhibin pro-{alpha}C (Fig. 1A) and bovine 32-kDa inhibin (Fig. 1B).



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  		FIG. 1. Validation of the ELISA systems for equine samples. Different dilutions of equine fetal plasma ({circ}) and equine ovarian homogenates ({square}) produced dose-response curves that were parallel to the standard curves () produced with human inhibin pro-{alpha}C (A) and bovine 32-kDa inhibin (B)

Plasma Concentrations of ir-Inhibin, Inhibin Pro-{alpha}C, and Inhibin A in Fetuses and Dams

Concentrations of ir-inhibin (Fig. 2A), inhibin pro-{alpha}C (Fig. 2B), and inhibin A (Fig. 2C) in peripheral plasma of fetuses and dams were not different among replicates between 100 and 150 days, 150 and 200 days, and 200 and 250 days of gestation. Therefore, data were combined to represent plasma concentrations at 100–150 days (n = 3), 150–200 days (n = 8), and 200–250 days (n = 4) of gestation. Comparison between fetuses and dams showed that plasma concentrations of ir-inhibin, inhibin pro-{alpha}C, and inhibin A were significantly (P < 0.05) higher in female fetuses than in dams at all stages of gestation examined. However, the concentrations of these inhibins were not different among the three different periods either in fetal or maternal circulation except that plasma concentrations of inhibin pro-{alpha}C in fetuses at Days 200–250 of gestation were significantly (P < 0.05) reduced compared with those at Days 100–150 and 150–200 of gestation.



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  		FIG. 2. Plasma concentrations (means ± SEM) of ir-inhibin (A), inhibin pro-{alpha}C (B), and inhibin A (C) in fetuses (open bars) and dams (solid bars) at Days 100–150 (n = 3), Days 150–200 (n = 8), and Days 200–250 (n = 4) of gestation. *P < 0.05: significance of differences between the fetuses and dams

Ovarian Concentrations of ir-Inhibin, Inhibin Pro-{alpha}C, and Inhibin A in Fetuses

High concentrations of all three inhibin forms were detected in the fetal ovarian homogenates (Fig. 3). Both ir-inhibin (Fig. 3A) and inhibin A (Fig. 3C) concentrations in the ovary tended to decrease as pregnancy advanced. On the other hand, ir-inhibin (Fig. 3D) and inhibin A (Fig. 3F) levels per ovary tended to increase toward late gestation, reflecting a massive growth of fetal ovaries. In contrast, inhibin pro-{alpha}C levels per gram of tissue (Fig. 3B) or per ovary (Fig. 3E) significantly (P < 0.05) increased from 100–150 to 150–200 days of gestation, followed by a significant (P < 0.05) reduction at Days 200–250 of gestation. Concentrations of inhibins in maternal ovaries and placenta between Days 100 and 250 of gestation were undetectable.



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  		FIG. 3. Ovarian concentrations (means ± SEM) of ir-inhibin (A), inhibin pro-{alpha}C (B), and inhibin A (C) and ovarian content (per ovary) of ir-inhibin (D), inhibin pro-{alpha}C (E), and inhibin A (F) in equine fetuses at Days 100–150 (n = 3), 150–200 (n = 8), and 200–250 (n = 4) of gestation. The bars with different letters were significantly (P < 0.05) different

Localization and Expression of Inhibin {alpha} and Inhibin/Activin ßA and ßB Subunits in Fetal Ovaries

During Days 100–250 of gestation, interstitial cells occupied approximately 90% of fetal ovaries (Fig. 4A). At Days 100, 150, 200, and 250 of gestation, immunostaining of the inhibin {alpha} (Fig. 4B) and inhibin/activin ßA (Fig. 4C) and ßB (Fig. 4D) subunits was detectable in interstitial cells of fetal ovaries. Immunostaining of the inhibin {alpha} and inhibin/activin ßA and ßB subunits of fetal ovaries tended to be most intense at around 200 days of gestation.



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  		FIG. 4. Photomicrographs of histological sections (5 µm) from equine fetal ovaries at Day 200 of gestation. Tissue sections were immunostained for the three inhibin subunits. A tissue section stained with hematoxylin and eosin is given in A. Tissue section incubated with respective antibody showed immunopositive staining for the inhibin {alpha} (B) and inhibin/activin ßA (C) and ßB (D) subunit proteins in interstitial cells of enlarged ovary, whereas the sections incubated with normal rabbit serum (E) or normal mouse serum (F) instead of primary antibody did not show immunopositive reaction. Bar = 50 µm

Expression of inhibin {alpha} and inhibin/activin ßA subunit mRNA (Fig. 5A and 5B) confirmed immunoreactivity in fetal ovaries at 200 days of gestation. Expression of inhibin {alpha} and inhibin/activin ßA subunit mRNAs were observed in interstitial cells of the fetal ovary at 200 days of gestation.



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  		FIG. 5. Expression of inhibin {alpha} (Fig. 4A) and inhibin/activin ßA subunit (Fig. 4A) mRNAs in interstitial cells of equine fetal ovary at 200 days of gestation. Both inhibin {alpha} and inhibin/activin ßA subunit mRNAs were observed in interstitial cells. Sense controls for inhibin {alpha} (Fig. 4C) and inhibin/activin ßA (Fig. 4C) subunits were negative. Bar = 50 µm


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study clearly demonstrated that peripheral plasma of equine female fetuses contained large amounts of ir-inhibin, inhibin pro-{alpha}C, and inhibin A. The inhibin levels are remarkably higher as compared with those of pregnant and cyclic mares [15–17, 24] and stallions [33–36]. The inhibin subunits, namely inhibin {alpha} and inhibin/activin ßA and ßB subunits, were mainly localized to the interstitial cells of fetal ovaries from mid- to late gestation. Expression of inhibin {alpha} and inhibin/activin ßA subunit mRNAs were also observed in the interstitial cells of fetal ovary at 200 days of gestation. It was also revealed in the present study that concentrations of inhibins in both maternal ovaries and placenta were undetectable, which confirmed our previous results [23]. Yamanouchi et al. [37] did not detect the expression of inhibin {alpha} subunit mRNA in the placenta of mares. Thus, the present results in conjunction with previous findings strongly suggest that the source of circulating inhibins in equine female fetuses is fetal ovaries and is not of maternal origin.

Circulating concentrations of ir-inhibin in both male and female ovine fetuses have been reported to be higher than those in maternal circulation [17, 19]. It was reported that gonadectomy in the fetus caused a rapid decline in circulating ir-inhibin levels, suggesting that the gonads were the primary source of circulating ir-inhibin [19]. The equine fetal gonads of both sexes have attracted much interest because of the remarkable enlargement in size as a result of massive number of interstitial cells between the third and ninth months of gestation [38, 39]. It is also known that the size of the equine fetal gonads during the second half of gestation (Days 200–250) is larger than that of the maternal ovaries [38, 40, 41]. The size of fetal gonads decreases after Day 250 of gestation, and the gonadal weight of newborn foal is approximately one tenth compared with that of fetal gonad at its maximum size [40]. Thus, our present results confirm the previously reported findings that fetal gonads are heavier than maternal gonads toward 250 days of gestation in the mare. Histologically, the interstitial cells are similar in fetal ovaries and testes [41, 42]. The ovary, but not testis, has a white cortical zone containing the germ cells and primordial follicles, and a thin germinal area is located on the ventral aspect of the ovary [43]. The enlarged gonads consist of approximately 90% interstitial cells [40, 43]. The present results clearly demonstrated that localization of inhibin {alpha} and inhibin/activin ßA and ßB subunits and expression of inhibin {alpha} and inhibin/activin ßA subunits were detectable in interstitial cells of fetal ovary. The results confirm previous findings that the inhibin {alpha} subunit mRNA [25] and protein [23] were detectable in interstitial cells of equine fetal ovaries. Thus, our results together with previous findings suggest that the interstitial cells of fetal ovaries are possible to produce dimeric inhibins and the major source of circulating inhibins in female fetuses is equine fetal ovaries. Although this massive enlargement of fetal gonads, comprising approximately 90% interstitial cells, occurs toward midgestation, its physiological significance or the mechanism responsible for the enlargement is not yet understood.

Expression and localization of the inhibin subunits in fetal ovaries have been reported in sheep [20], monkeys [22], humans [22], and cows [21]. In ovine fetal ovaries at or near full term, a number of granulosa cells of antral follicles expressed both the inhibin {alpha} subunit mRNA and peptide, but there was no detectable expression of the ßA subunit mRNA or peptide at any stage of gestation [20]. In contrast, in bovine fetal ovaries, the {alpha} and ßA subunit mRNAs have been observed in the cortical region of the ovary at an early stage of gestation and in the granulosa cells of antral follicles at a later stage of gestation [21]. In the fetal rhesus monkey ovary, the granulosa cells surrounding the oocyte were immunostained for all three inhibin subunits at late gestation; however, neither thecal nor interstitial cells showed any immunopositive reaction [22]. These authors also observed a weak inhibin/activin ßA subunit immunostaining in a few primary follicles of human fetal ovaries at midgestation. In the present study, we found that the interstitial cells of enlarged equine fetal ovaries produced all three subunits such as the inhibin {alpha} and inhibin/activin ßA and ßB subunits. We conclude that the interstitial cells of enlarged equine fetal ovaries secrete large amounts of dimeric (bioactive) inhibins. These inhibins may play some physiological roles in the development of fetal gonads, including the enlargement of ovaries.


   ACKNOWLEDGMENTS
 
The authors are grateful to N. Ling (Neuroendocrine Inc., San Diego, CA) for providing [Tyr30] porcine inhibin-chain (1-30)-NH2 conjugated to rabbit serum albumin.


   FOOTNOTES
 
1 This work was supported by a grant-in-aid from the Equine Research Institute of the Japan Racing Association. Back

2 Correspondence: Kazuyoshi Taya, Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, 3–5–8, Saiwai-cho, Fuchu, Tokyo 183-8509, Japan. FAX: 81 42 367 5767; taya{at}cc.tuat.ac.jp Back

Received: 17 January 2002.

First decision: 19 February 2002.

Accepted: 3 July 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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