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BOR - Papers in Press, published online ahead of print March 17, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.027789
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BIOLOGY OF REPRODUCTION 71, 236–243 (2004)
DOI: 10.1095/biolreprod.104.027789
© 2004 by the Society for the Study of Reproduction, Inc.

Reproductive Technology

A New Alternative Method for Superovulation Using Passive Immunization Against Inhibin in Adult Rats1

Harumichi Ishigame3,4, Mohamed S. Medan3,5, Gen Watanabe3,6, Zhanguan Shi6, Hisahi Kishi3, Koji Y. Arai4, and Kazuyoshi Taya2,3,6

Laboratory of Veterinary Physiology,3 Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan Department of Tissue Physiology,4 Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan Department of Theriogenology,5 Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt Department of Basic Veterinary Sciences,6 The United Graduate School of Veterinary Sciences, Gifu University, Gifu 501-1193, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study was undertaken to investigate the effects of passive immunoneutralization of endogenous inhibin on ovulation rate and embryo development in vivo and in vitro to establish a new alternative superovulation method in the adult rat. Female adult rats of Wistar strain were superovulated with a single injection of inhibin antiserum (inhibin-AS; 100 or 400 µl) or an injection of 20 IU eCG followed by an injection of 10 IU hCG. Untreated animals served as controls. Embryos were collected from oviducts or uteri on Days 1–5 of pregnancy, and the number of embryos and implantation sites were observed. On Day 1 of pregnancy, the two-cell-stage embryos were cultured and embryos from the 100-µl inhibin-AS group and the control group were transferred to recipient females to determine developmental competence. There were no significant differences between groups in fertilization rate. The numbers of normal embryos in the inhibin-AS-treated groups were significantly higher than the control and the eCG-hCG-treated groups throughout Days 1–4 of pregnancy. The number of implantation sites observed on Day 5 of pregnancy in the inhibin-AS-treated groups was significantly higher than both the control and the eCG-hCG-treated groups. Furthermore, the rate of blastocyst development in vitro in the inhibin-AS-treated groups and posttransfer viability in the 100-µl-inhibin-AS group were comparable with those of the control group. These results indicate that immunoneutralization of endogenous inhibin is a new practical alternative for induction of superovulation as a substitution for eCG-hCG method in the adult rat.

embryo, implantation, inhibin, ovary, ovulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In recent years, transgenic and cloning technology in animals is increasingly important for biotechnological and physiological studies. The rat is a prime animal model for studying human diseases, such as hypertension ENRfu [1], diabetes [2], and autoimmunity [3]. Transgenic rat technology also opens up interesting perspectives for transplantation research [4, 5], in which microsurgery is an essential procedure. Due to greater size, for continuous or repeated blood collection and surgical treatments, rats could be more practical models than mice. However, reproductive technologies in rats have not yet been established compared with those of mice. In this respect, the methods for the induction of superovulation for collection of large numbers of oocytes or zygotes are very important as a basic technology to develop these studies in rats. Although the successful multiple ovulation has been established in immature rats [6–8], it is rather difficult to induce superovulation in adult cyclic rats due to different reactions to hormonal treatment on various days of the ovarian cycle [9, 10]. So far, a practical method for use with adult rats is not yet available. Adult rats seem to be beneficial because of stable mating rates compared with immature rats, which enable us to obtain fertilized oocytes.

The most common method for induction of superovulation is treatment with a combination of equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). However, several studies have reported high rates of pre- and postimplantation losses in animals superovulated with eCG. The early developmental failure has been shown to be related to a hostile maternal environment [8], which is thought to be a result of excessive follicular stimulation because of the inherent long biological half-life of eCG. High doses of eCG may have a detrimental effect on oocyte quality, such as the ability to be fertilized [11, 12], impaired development [13–17], and/or chromosomal abnormalities [18]. Therefore, we tried to find an alternative method for producing large numbers of viable oocytes and zygotes in adult rats.

Inhibin is an essential hormone regulating FSH secretion in various mammals [19] and is composed of disulfide-bonded {alpha} and ß subunits. Combinations of an {alpha} subunit with either ß subunits ({alpha}/ßA or {alpha}/ßB) form inhibin molecules with potent bioactivity for inhibiting FSH secretion. In previous studies, a negative relationship between plasma concentration of FSH and inhibin has been established [20– 26]. Multiple ovulations have been induced successfully by passive immunization against endogenous inhibin in several species such as mice [27], rats [28], hamsters [29], cows [30, 31], mares [32], and goats [33]. Furthermore, several studies have shown that oocytes superovulated with immunization against inhibin have the ability to develop normally [27, 31, 34, 35], suggesting that inhibin immunization could be applicable to a wide range of animal species. Although Rivier and Vale [28] reported that increased numbers of pups were obtained after immunoneutralization of inhibin in rats, detailed analysis on embryo quality during peri-implantation was not conducted.

The aim of this study was to examine the efficacy of immunoneutralization of endogenous inhibin in the adult rats on ovulation rate and the competence of embryo development in vivo and in vitro to be used in reproductive-developmental research.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Adult cyclic female rats of the Wistar strain (2–3 mo of age) were used. They were kept in a room with controlled illumination (14L:10D, lights-on at 0500 h) and temperature (25 ± 2°C), with access to food and water ad libitum. Vaginal smears were checked daily and only rats with at least two consecutive 4-day cycles were used. The experimental protocol was approved in accordance with the Guide for the Care and Use of Laboratory Animals prepared by the Tokyo University of Agriculture and Technology.

Inhibin Antiserum

The inhibin {alpha} subunit antiserum (inhibin-AS) was obtained from castrated goats immunized against [Tyr30]-inhibin {alpha}(1-30)-NH2 conjugated to rabbit serum albumin. This conjugate was kindly provided by Dr. N Ling (Neuroendocrine Biosciences, Inc., San Diego, CA). The titer of the antiserum was determined as in our previous reports [36]. The serum used in the present experiment had a titer of 1:1 000 000 as defined by final dilution of the antiserum required to bind 50% of added 125I-labeled bovine 32-kDa inhibin. The in vivo efficiency of the antiserum was ensured by an increase in plasma concentrations of FSH after an i.v. injection of the antiserum, as described previously [37].

Effects of Treatment with Inhibin-AS and eCG-hCG on Concentrations of Hormones During Estrous Cycle

Female rats were administered a single i.v. injection of different doses of inhibin-AS (100 or 400 µl per animal) at 1100 h on the day of metestrus into the jugular vein under light ether anesthesia or an i.p. injection of 20 IU eCG (Sigma Chemical Co., St. Louis, MO). After 54 h (at 1700 h in proestrus), rats injected with 20 IU eCG were given an injection of 10 IU hCG (Sankyo Zoki Co. Ltd., Tokyo, Japan). Untreated animals served as controls. The animals were decapitated every 6 or 12 h after treatment and trunk blood samples were collected into heparinized centrifuge tubes. Blood samples were centrifuged immediately at 1700 x g for 30 min at 4°C. Plasma was separated and stored at –20°C until assayed for FSH, LH, ir-inhibin, estradiol-17ß, and progesterone.

Effects of Treatment with Inhibin-AS and eCG-hCG on Fertilization and Early Embryo Development In Vivo

After the treatment with superovulation protocol described above, the individual female rats were mated with a fertile male rat of the same strain on the evening of proestrus (after hCG administration in the eCG-treated group). The next morning, rats were examined for the presence of a vaginal plug or spermatozoa in the vagina. This day was designated Day 0 of pregnancy. At 0900 h on Days 1–4 of pregnancy, animals were killed by decapitation; blood samples, oviducts, and uteri were collected. Blood samples were centrifuged immediately and plasma was separated and stored at –20°C until assayed for estradiol-17ß and progesterone. Oviducts and uteri were separated at the utero-tubal junction and differentially flushed with saline. The recovered embryos were counted under a dissecting microscope and scored for the occurrence of fertilization. Embryos with regular blastomeres and intact zonae pellucidae were classified as embryos with normal morphology, whereas embryos with irregular blastomeres or those degenerating or fragmented were classified as embryos with abnormal morphology. Embryos showing compaction and blastocoele cavity were classified as morula and blastocyst, respectively. To examine the number of implantation sites, animals were injected i.v. with a 1% (w/ v) pontamine blue solution (500 µl) under ether anesthesia at 0900 h on Day 5 of pregnancy to extravasate into implantation area in the endometrium, then killed by decapitation 15 min after administration.

Effects of Treatment with Inhibin-AS on Early Embryo Development In Vitro

At 0900 h on Day 1 of pregnancy, embryos were collected by flushing the excised oviducts with mR1ECM [38] through the fimbrial opening. Only normal two-cell embryos were used. They were washed three times with mR1ECM and then placed (10–20 embryos) in 20-µl droplets of the respecting culture medium, which had previously been covered with silicon oil (Aldrich Chemical Co. Inc., Milwaukee, WI) and then cultured for 96 h at 37°C, 5% CO2 in air, 95% relative humidity.

Effects of Treatment with Inhibin-AS on Postimplantation Viability of Embryos after Transfer to Pseudopregnant Rats

Two-cell-stage embryos that recovered from control and 100-µl-inhibin-AS-treated rats were transferred into the oviducts of Day 0 of pseudopregnant Wistar recipients with a fine glass pipette under microscopic visualization. The pseudopregnancy of the females was induced by mating with vasectomized males. For embryo transfer, 3- to 4-mo-old rats were anesthetized with an i.p. injection of 3.5 mg/100 g pentobarbital sodium (Dainippon Pharmaceutical Co., Ltd, Japan) between 0900 h and 1200 h. Seven to eight embryos from both control and 100-µl-inhibin-AS-treated groups were transferred to contralateral oviducts of pseudopregnant animals.

RIA of FSH, LH, ir-Inhibin, Estradiol-17ß, and Progesterone

Plasma concentrations of FSH and LH were measured using an NIDDK (Bethesda, MD) RIA kits for rat FSH and LH. Iodinated preparations were rat FSH-I-5 and LH-I-5. The antisera used were anti-rat FSH-S-11 and anti-rat LH-S-11. Results were expressed as rat FSH RP-2 and rat LH RP-3. The intra- and interassay coefficients of variations were 4.8% and 11.4%, respectively, for FSH and 5.4% and 6.9%, respectively, for LH. Plasma concentrations of immunoreactive (ir-) inhibin were measured using rabbit antiserum against bovine inhibin (TNDH-1) and 125I-labeled 32-kDa bovine inhibin, as described previously [39]. Results were expressed in terms of 32-kDa bovine inhibin. The intra- and interassay coefficients of variation were 8.8% and 14.4%, respectively. Concentrations of estradiol-17ß and progesterone in plasma were determined by double-antibody RIA systems using 125I-labeled radioligands, as described previously [40]. Antisera against estradiol-17ß (GDN 244) and progesterone (GDN 377) provided by Dr G.D. Niswender (Colorado State University, Fort Collins, CO) were used. The intra- and interassay coefficients of variations were 4.8% and 11.4%, respectively, for estradiol-17ß and 5.4% and 6.9%, respectively, for progesterone.

Changes in the Concentration of Inhibin-AS in Peripheral Plasma

The changing pattern of inhibin-AS in the plasma after the injection was determined as described previously [36]. Briefly, 125I-labeled inhibin was added to dilutions of plasma sample. After equilibration overnight at 32°C, precipitation was induced by the addition of polyethylene glycol, and radioactivity in the precipitate was measured. Results were expressed as nanoliter equivalents of undiluted standard inhibin-AS/ml plasma. The intra- and interassay coefficients of variation were 1.8% and 3.9%, respectively.

Statistics

Mean values (±SEM) were calculated and analyzed using two-way ANOVA. Duncan multiple-range test was used for detection of significant differences using the SAS computer package [41]. The rate of implantation sites was calculated as a percentage of the total number of embryos recovered on Day 1 of pregnancy. The chi-square test was used to assess if there were any differences in the mating rate, fertilization rate, rate of normal embryos, percentage of embryos recovered from oviduct and uterus, rate of implantation sites, and percentage of viable fetuses. A probability of P < 0.05 was considered to be significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Changes in Plasma Levels of FSH, LH, ir-Inhibin, Estradiol-17ß, and Progesterone in Inhibin-Immunized and eCG-hCG-Treated Groups During Estrous Cycle

Plasma levels of inhibin-AS gradually decreased after treatment, as shown in Figure 1. However, the values of inhibin-AS decreased to undetectable levels within 48 h in the group treated with 100 µl and remained high throughout the experiment in the group treated with 400 µl. Plasma levels of FSH significantly increased to the preovulatory surge levels of the control group within 12 h of the administration of 100 µl or 400 µl inhibin-AS (Fig. 2A). The increased levels of plasma FSH were similar in animals treated with either 100 µl or 400 µl until 48 h after administration. However, the preovulatory surge of FSH was higher in animals treated with 400 µl inhibin-AS compared with those treated with 100 µl. In contrast, plasma concentrations of FSH were slightly lower in eCG-hCG-treated animals than in control ones. A slight increase in plasma LH concentrations was observed in inhibin-AS-treated groups until 48 h after injection in animals treated with 100 µl inhibin-AS (Fig. 2B). The LH surge was lower in animals treated with inhibin-AS compared with the control group. On the other hand, plasma concentrations of LH increased significantly after the administration of eCG compared with the other groups and no preovulatory LH surge was observed in the group treated with eCG-hCG. Plasma levels of ir-inhibin significantly increased within 12 h after administration of eCG in comparison with those of the control group (Fig. 2C), reaching a peak level at 1700 h in proestrus and suddenly declining at 2300 h in proestrus.



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  		FIG. 1. Changing pattern of inhibin-AS in peripheral plasma of adult cyclic rats after administration of 100 µl (white circle) or 400 µl (white triangle) inhibin-AS on the day of metestrus. Values represent mean ± SEM for five or eight rats



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  		FIG. 2. Changes in plasma levels of FSH (A); LH (B); ir-inhibin (C); estradiol-17ß (D); and progesterone (E) in adult cyclic rats treated with 100 µl inhibin-AS (white diamond), 400 µl inhibin-AS (black square), eCG-hCG (white triangle) on the day of metestrus or control group (white circle). Each point represents mean ± SEM of 5–8 rats. Values with different superscripts are significantly different (P < 0.05, Duncan multiple-range test)

Plasma levels of estradiol-17ß showed a rapid increase in inhibin-AS-treated groups compared with other groups. However, the peak level of estradiol-17ß in the eCG-hCG-treated group was extremely high in comparison with inhibin-AS-treated and control groups (Fig. 2D). In addition, changes in plasma concentrations of progesterone were similar in inhibin-AS-treated and control groups. However, progesterone levels in the eCG-hCG-treated group showed a significant increase 12 h after injection of eCG and continued high in diestrus and early proestrus. Thereafter, plasma concentrations of progesterone were further increased within 6 h after treatment with hCG (Fig. 2E).

Effects of Treatment with Inhibin-AS and eCG-hCG on Embryo Production, Fertilization, and Embryo Development In Vivo

The percentage of mating rats was 97.3% in the group treated with 100 µl inhibin-AS and 100% in all other groups, showing no significant difference. Female rats that did not mate were not included in the present study. The number of embryos and the percentage of normal embryos for each treatment group are shown in Table 1. Among the embryos recovered on Day 1 of pregnancy, 97.5%, 96.5%, 96.5%, and 93.6% were fertilized in the control, eCG-hCG, and 100-µl-inhibin-AS- and 400-µl-inhibin-AS-treated groups, respectively. The rate of fertilization was not statistically different among groups. The number of recovered embryos in inhibin-AS-treated groups was significantly higher than either control or eCG-hCG-treated groups throughout Days 1–4 of pregnancy. Also, the number of recovered embryos in the group treated with eCG-hCG was significantly higher than that of the control group throughout Days 1–3 of pregnancy. These differences were also observed in the number of morphologically normal embryos in both eCG-hCG- and inhibin-AS-treated groups. However, the percentage of normal embryos in both inhibin-AS- and eCG-hCG-treated groups were considerably lower compared with that in the control group on Days 3 and 4 of pregnancy. In addition, there was a reduction in the number of recovered embryos in inhibin-AS- (100 µl and 400 µl) and eCG-hCG-treated groups on Day 4 of pregnancy compared with the respective values on Day 1 of pregnancy.


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  		TABLE 1. Effects of treatment with inhibin-AS or eCG-hCG administered on the day of metestrus in adult cyclic rats on the number of embryos and the number of normal embryos recovered on Days 1–4 of pregnancy

Distributions of embryos between oviducts and uterine horns are shown in Table 1. Whereas embryos recovered from uterus in rats treated with 400 µl inhibin-AS were first observed on Day 1 of pregnancy, the remaining groups were observed on Day 3 of pregnancy. The percentage of embryos recovered from uteri increased significantly on Day 3 of pregnancy in rats treated with inhibin-AS and eCG-hCG. In all animals, embryos were recovered only from the uterus on Day 4 of pregnancy. The number of implantation sites observed on Day 5 of pregnancy in groups treated with inhibin-AS (100 µl and 400 µl) was more than both eCG-hCG-treated and control groups (Table 2). However, the rates of implantation sites in eCG-hCG- (69.9%) and inhibin-AS-treated rats (100 µl: 55.2%, 400 µl: 43.3%) were significantly lower in comparison with control rats (91.3%).


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  		TABLE 2. The number of implantation sites on Day 5 of pregnancy of adult rats treated with inhibin-AS or eCG-hCG

Changes in Plasma Levels of Estradiol-17ß and Progesterone During Early Pregnancy

There was a significant increase in the plasma levels of estradiol-17ß in the inhibin-AS-treated groups when compared with those of the control group (Fig. 3A). In the eCG-hCG-treated group, estradiol-17ß was relatively higher than those of the control group and the difference was significant at Day 3 of pregnancy. Furthermore, a marked increase in plasma concentrations of progesterone was noted in the inhibin-AS-treated groups throughout Days 1–4 of pregnancy as compared with the control (Fig. 3B). In the eCG-hCG-treated group, only a slight elevation in progesterone level was noticed compared with the control group.



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  		FIG. 3. Changes in plasma levels of estradiol-17ß (A) and progesterone (B) during early pregnancy in adult rats treated with 100 µl inhibin-AS (white diamond), 400 µl inhibin-AS (black square), eCG-hCG (white triangle) on day of metestrus or control group (white circle). Each point represents the mean ± SEM of 5–8 rats. Values with different superscripts are significantly different (P < 0.05, Duncan multiple-range test)

Effect of Inhibin-AS on Embryo Development In Vitro

The number of two-cell-stage embryos recovered and the rate of blastocyst development are shown in Table 3. The number of two-cell-stage embryos in the groups treated with either inhibin-AS or eCG-hCG was significantly higher than that of the control group. Moreover, the number of two-cell-stage embryos in groups treated with inhibin-AS was significantly higher than that of the eCG-hCG-treated group. There was no significant difference among groups in the rate of blastocyst development.


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  		TABLE 3. Effects of inhibin-AS or eCG-hCG administered on the day of metestrus in adult cyclic rats on production of blastocysts and rate of blastocysts development in vitro.a

Effect of Inhibin-AS on Postimplantation Viability of Two-Cell-Stage Embryos after Transfer to Pseudopregnant Rats

Postimplantation development of embryos retrieved from the group treated with 100 µl inhibin-AS or control group were assessed. Twelve pseudopregnant recipient female rats were used in each group. A total of 95 two-cell-stage embryos from the inhibin-AS-treated group produced 73 implantation sites (fetuses plus resorption sites) compared with 71 implantation sites out of 94 embryos from the control group. Moreover, there were no significant differences between the two groups in the number or weight of the viable fetuses, as shown in Table 4.


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  		TABLE 4. Effect of inhibin-AS administered on the day of metestrus in adult cyclic rats on postimplantation viability of two-cell-stage embryos examined 20 days after transfer to pseudopreganant rats.a


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of the present study clearly showed that adult rats can be effectively superovulated to produce more than a threefold increase in ovulation rate over control animals and a twofold increase over eCG-hCG-treated animals through passive immunization against inhibin. Only a single injection of inhibin-AS is needed to induce superovulation in adult rats, making it more practical and efficient compared with the previous studies, such as eCG-hCG [9, 42], anterior pituitary implants [43], FSH preparations [10], and recombinant human FSH [44].

In this study, immunoneutralization of endogenous inhibin in the adult rats increased plasma concentration of FSH. Also, plasma levels of estradiol-17ß significantly increased in the rats treated with inhibin-AS. These findings suggest that a high level of endogenous FSH stimulates the wave of follicular development and results in production of a large amount of estradiol-17ß, which induces the LH surge by positive feedback effect to the hypothalamus and pituitary axis, leading to induction of superovulation. Increased FSH levels in inhibin-AS-treated rats indicate that endogenous inhibin is a primary factor in the control of species-specific ovulation rate, mainly through the control of FSH secretion, as described previously in many species [20, 21]. In addition, treatment with eCG resulted in a dramatic increase in the secretion of inhibin and estradiol-17ß. These increases were accompanied by a decrease in plasma levels of FSH as observed previously [45–47]. Our results indicate that stimulation of follicular development by administration of eCG was thought to induce an increase in plasma inhibin, which in turn suppress FSH secretion.

While the plasma levels of progesterone were not affected after administration of inhibin-AS, it increased dramatically in eCG-hCG-treated rats as compared with control and inhibin-AS-injected rats. It was demonstrated that administration of progesterone consistently postponed the LH surge in 4-day cyclic rats [48]. These observations suggest that extremely high levels of plasma progesterone probably prevent induction of the LH surge in eCG-hCG-treated rats. These findings also suggest that the normal concentrations of progesterone could be able to induce the gonadotropin surge at the expected time without an additional hCG injection after treatment with inhibin-AS.

In the present study, basal plasma LH levels increased after immunoneutralization of endogenous inhibin as reported previously [29, 37, 49]. In the eCG-hCG-treated group, plasma concentrations of LH increased significantly during the experimental period. This may be attributed to the cross reaction of exogenous eCG in the present LH RIA, which is in agreement with a previous study [50].

In the present study, a normal mating rate was encountered in the animals given inhibin-AS and the embryos collected from the animals treated with inhibin-AS could be fertilized equally as oocytes retrieved from nonstimulated control rats. In addition to providing a high yield of fertilization oocytes, high recoveries of morphologically normal developing embryos occurred at all days examined in rats treated with inhibin-AS compared with the control and the eCG-hCG-treated animals. Rivier and Vale [28] reported that a large number of implantation sites were observed after immunization against inhibin, supporting our results that embryos superovulated with inhibin-AS are capable of implanting. However, the marked decline in the number of implantation sites observed on Day 5 in the inhibin-AS-treated rats may be due to spacing problems in the uterus with this larger-than-normal number of embryos.

There was a partial loss in embryos in inhibin-AS-treated groups between Days 3 and 4 of pregnancy, which was also observed in the eCG-hCG-treated group. It has been proposed that embryo loss after superovulation with eCG is due to acceleration of embryo transport within the reproductive tract. This has been associated with a high level of postovulatory circulating estradiol-17ß. Estradiol-17ß is known to stimulate oviductal motility and accelerate embryo transport in rats [51, 52]. In addition to the accelerated transport of embryos recorded in the present study, there were higher levels of plasma estradiol-17ß in inhibin-AS-treated animals compared with control animals, indicating that maternal environment in superovulated animals was different from suitable normal environment for developing embryos. It appears likely that the elevated circulating estradiol-17ß levels and resultant embryo loss were attributable to excessive follicular stimulation persisting during the preimplantation period. Exposure to the stimulated oviductal and uterine environment is detrimental for embryonic implantation and fetal viability [8, 11, 53]. It is known that estradiol receptor mRNA and its protein were identified in preimplantation mouse embryos [54], suggesting that estradiol could act directly on the embryos. Effects of estradiol-17ß on the preimplantation embryos have been shown to be detrimental in vitro in the rat [55, 56]. These findings suggest that the losses of oocytes during the preimplantation period observed in the present study in rats treated with inhibin-AS may have been due to not only expulsion from the reproductive tracts but also degeneration beyond recognition within the tracts. The high number of implantation sites observed in animals treated with inhibin-AS supports this interpretation.

To evaluate the influence of the maternal environmental factor, which is thought to be detrimental in development of peri-implantation embryos in superovulated animals, we examined the embryonic developmental competence by in vitro culture and embryo transfer experiments. The superovulated two-cell embryos collected from the animals given inhibin-AS were able to develop normally to the blastocyst stage. Furthermore, the percentage of embryos in the animals treated with 100 µl inhibin-AS observed in 20-day-old pregnancies was similar to that of nonstimulated animals. These results indicate that oocytes superovulated with inhibin-AS have normal developmental competence. These results also suggest that the cause of adverse effects on embryo developmental competence observed in vivo may not be attributed to defects in the oocytes induced to superovulate using inhibin-AS, but rather to the maternal environment. Previous studies in mice and rats showed abnormalities in preimplantation embryo development after eCG stimulation [13, 16]. However, embryos obtained from eCG-treated and nonstimulated animals had equal developmental potential after embryo transfer [57, 58], supporting our present results. On the other hand, a decrease in viability posttransfer of embryos from eCG-stimulated hamsters was observed [59]. These discrepancies may be explained by the influence of various gonadotropins on preimplantation embryo development being dependent upon a number of factors, including species and/or strain, dosage, age, or weight of the animals used and the exposure time in the reproductive tract [42, 60, 61]. Further studies are needed to examine the effectiveness of inhibin-AS in immature rats or any other strains that are known to be poor responders to eCG-hCG.

In conclusion, the oocytes induced to superovulate using passive immunization of endogenous inhibin {alpha} subunit have normal developmental competence. Therefore, immunization of rats against inhibin could be a new alternative for induction of superovulation to be used in reproductive-developmental research instead of eCG-hCG more commonly used.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. A.F. Parlow and the Rat Pituitary Hormone Distribution Program (NIDDK, NIH, Bethesda, MD) for providing RIA materials, to Dr. G.D. Niswender (Animal Reproduction and Biotechnology, Colorado State University, Fort Collins, CO) for providing antisera to estradiol-17ß (GDN 244) and progesterone (GDN 377), and to Dr. N. Ling (Neuroendocrine Inc., San Diego, CA) for providing [Tyr30]-inhibin {alpha} (1-30).


   FOOTNOTES
 
1 Supported by a Grant-in-Aid for COE (E-1) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. 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: 26 January 2004.

First decision: 23 February 2004.

Accepted: 4 March 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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