Maintenance of functional corpora lutea in androgenized female rats treated with OMSG

Rats were androgenized by injection of 50 micrograms testosterone propionate on the 5th day after birth and when adult were treated with 5 i.u. PMSG; some of the animals were mated. Serum was obtained daily and the concentrations of progesterone, 20 alpha-dihydroprogesterone and prolactin, estimated by radioimmunoassays, were compared to values found for mated, but not ovulating, androgenized females and those for normal pregnant females. Ovulation and luteinization of follicles occurred. The concentration of progesterone increased after the injection of PMSG and remained elevated for at least 10 days; mating did not alter the progesterone levels. The concentration of 20 alpha-dihydroprogesterone was also elevated but the ratio of the level of progesterone to this steroid was generally greater than unity. Prolatin levels were elevated in the rats which ovulated. It is concluded that the corpora lutea induced in androgenized females by PMSG are functional and maintained.     

Relationship between the production capacity of ovarian 13,14-dihydro-prostaglandin F2-alpha and the process of ovulation in immature female rats pretreated with gonadotropin

The purpose of this work was to investigate the effects of gonadotropin on the production capacity of ovarian 13,14-dihydro-prostaglandin F2-alpha (13,14H2-PGF2 alpha) and whether or not this capacity had any relation to the process of ovulation in rat. To induce the first ovulation, immature rats were injected subcutaneously with PMSG (5 IU/rat) at 8:00 at 26 days of age and some of these rats were followed by an intraperitoneal injection of hCG (10 IU/rat) at 57 hrs after PMSG treatment. The 13,14H2-PGF2 alpha production capacity was unchanged as compared with vehicle control until 57 hrs after PMSG treatment. However, the capacity showed a striking increase at 60 hrs after PMSG treatment. A maximal increase of about 7 fold was observed at 9 hrs after hCG injection just before ovulation. The production capacity of the Graafian follicle (GF) and the part (WO-GF) of the whole ovary (WO) from which the GF is removed at 2:00 on day 29 and the capacity of early corpus luteum at 8:00 on day 29 was greater than that of GF and WO-GF at 0:00 on day 29. These results suggest that the 13,14H2-PGF2 alpha production capacity in rat ovary is regulated by gonadotropin and is closely associated with the process of ovulation.     

Fertilizability of Superovulated Eggs by Estrous Stage-independent PMSG/hCG Treatment in Adult Wistar-Imamichi Rats

We investigated the fertilization and developmental ability of superovulated eggs obtained from adult Wistar-Imamichi (WI) rats, by using pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) treatment. Female WI rats, 11–13 weeks of age, were divided into four groups by estrous stage (metestrus [ME], diestrus [DE], proestrus [PE], or estrus [E]). PMSG (150 IU/kg) and hCG (75 IU/kg) were injected at an interval of 48 or 55 h and the female rats were mated with mature male rats. The ovulated eggs were collected 20, 24, and 27 h after hCG injection. Regardless of the estrous stage at the time of PMSG injection, the treated rats mated and ovulated similar to the untreated spontaneously ovulated rats (S group). Although the proportion of fertilized eggs in the E- and PE-treated groups was less than the S group 20 h after hCG injection, the proportion was not different among all treated and S groups 24 h after hCG injection. The proportion of fertilized eggs using in vitro fertilization and the proportion of offspring obtained from 2-cell stage embryo transfer did not differ among the treated and S groups. In comparison with PMSG/hCG-treated immature rats, mating and ovulation rate of adult rats were significantly higher. The proportion of fertilized eggs obtained from mated rats did not differ between immature and adult rats. These results demonstrate that adult WI rats are good egg donors for reproductive biotechnological studies using unfertilized or fertilized eggs.     

Selective release of follicle-stimulating hormone and luteinizing hormone by 5 alpha-dihydroprogesterone and 3 alpha, 5 alpha-tetrahydroprogesterone in pregnant mare's serum gonadotropin-primed immature rats exposed to constant light

The effects of 5 alpha-dihydroprogesterone (5 alpha-DHP) and 3 alpha, 5 alpha-tetrahydroprogesterone (3 alpha, 5 alpha-THP) on follicle-stimulating hormone (FSH) and luteinizing hormone (LH) release were examined in the pregnant mare's serum gonadotropin (PMSG)-primed immature female rat (8 IU PMSG at 28 days of age) maintained in constant light. Control rats kept in 14L:10D conditions exhibited proestrous-like surges of LH and FSH release with peak levels attained at 1800 h on the second day after PMSG treatment. In rats exposed to constant light, the PMSG-induced surges of LH and FSH were not only delayed until 1000 h on the third day after PMSG, resulting in a delay in ovulation, but were also significantly attenuated when compared to the gonadotropin surges that occurred on Day 2 in rats kept under normal light-dark conditions. The administration of 5 alpha-DHP significantly enhanced the release of FSH at 1000 h on Day 3 when compared to constant light-exposed controls, but had no effect on LH. Treatment with 3 alpha, 5 alpha-THP selectively potentiated the release of LH at 1000 h on Day 3 and had an attenuating effect on FSH release on Days 2 and 3. These observations confirm earlier findings in the immature ovariectomized estrogen-primed rat and suggest that 5 alpha-DHP and 3 alpha, 5 alpha-THP may have significant roles in the regulation of FSH and LH secretion.     

Comparison between PMSG- and FSH-induced superovulation for the generation of transgenic rats

Superovulation protocols using single injections of pregnant mare's serum gonadotropin (PMSG) or minipumps with follicle-stimulating hormone (FSH) were compared in immature Sprague-Dawley (SD) rats. We used the following criteria: total number of ova, rate of fertilization, in vitro embryo development, sensitivity of zygotes to the microinjection of foreign DNA into the pronucleus, and their in-vivo development after transplantation into the oviduct of a recipient. Female SD rats were stimulated with 15 IU PMSG or 10 mg FSH followed by the injection of human chorionic gonadotropin (hCG) at doses of 20 and 30 IU per female. After hCG administration, they were mated with males of the same strain and sacrificed on day 1 of pregnancy. The percentage of mated animals and the fertilization rate was similar in all groups. In rats given PMSG, the number of ovulated zygotes was hCG dose-dependent. In contrast, the dose of hCG did not influence the efficiency of superovulation in rats given FSH, which was equal to PMSG-treated rats at the optimal dose of hCG. The rates of in vitro blastocyst development (31.4 and 23.3%) and the resistance to microinjection into the pronucleus did also not differ significantly between zygotes of both studied groups. The proportion of offspring developing from microinjected zygotes after oviduct transfer (26.2 and 26.8%, respectively) and the rate of transgene integration per newborns (7.3 and 4.9%, respectively) was similar in both experimental groups. The results of this study demonstrate that superovulation of immature SD rats by PMSG is equally effective as FSH treatment and, thus, preferable for transgenic rat technology due to the lower costs and easier handling.     

Inhibition of ovulation in the gonadotropin-primed immature rat by exogenous prostaglandin E2.

In the past two decades there have been innumerable reports that prostaglandins (PGs) are essential for mammalian ovulation. However, we have recently found that a relatively low dose of 0.03 mg indomethacin (INDO) sc to PMSG/hCG-primed immature Wistar rats can significantly reduce ovarian PG levels without inhibiting the control ovulation rate of 60+ ova/rat (1-3). In view of this information, the present study was an effort to duplicate the earlier reports that PGs can reverse the "inhibitory" effect of INDO on ovulation. In control animals, which received PMSG and hCG only, the ovulation rate was 63.8 +/- 4.5 ova/rat. This rate was reduced to 4.1 +/- 1.1 ova/rat when the animals were injected with 1.0 mg INDO at 3 h after hCG. In no instance was this inhibition reversed when the animals were treated with 1.0 mg of PGE2 or PGF2 alpha, or a combination of both prostanoids in either a single dose at 3 h after hCG, or in 4x doses at 2-h intervals beginning at 3 h after hCG. Furthermore, in animals that did not receive INDO, the ovulation rate in PGE2-treated animals was reduced to 20.0 +/- 6.7 ova/rat, and in animals treated with PGE2 and PGF2 alpha (combined) it was reduced to 19.4 +/- 6.5 ova/rat. In summary, not only did the PGs fail to reverse the anti-ovulatory effect of INDO, PGE2 actually suppressed the ovulation rate.     

Physiological peripubertal activation of the ovary is not reproduced by pregnant mare serum gonadotropin (PMSG) administration

During the days preceding the first ovulation the ovary of the rat exhibits a remarkable increase in estradiol (E2) and progesterone (P) release in response to gonadotropins. No such increase is observed in the case of androgens (A, testosterone + dihydrotestosterone). The present experiments were undertaken to examine the possibility of reproducing these developmental events by stimulating the ovary with a gonadotropin that has substantial FSH-like activity. In vivo administration of pregnant mare serum gonadotropin (PMSG) to juvenile 29-day-old rats greatly increased the in vitro E2 and A response to human chorionic gonadotropin (hCG) measured 2 days later in the morning. The magnitude of the A response was significantly larger than that of ovaries from juvenile animals or rats in first proestrus. The E2 response was much greater than that of juvenile ovaries but similar to that of ovaries from late proestrous rats. In contrast, the P response to hCG was not enhanced by PMSG. In fact the response was similar to that of juvenile ovaries and markedly less than that of first proestrous rats. This decreased P response was not due to a greater conversion of P to its less active metabolite 20 alpha-hydroxy-4-pregnen-3-one (20 alpha-OH-P). The results suggest that PMSG enhances the E2 and A response of immature ovaries to hCG at the expense of that of P. Treatment of immature rats with PMSG may represent a useful model to study E2 release from preovulatory ovaries, but it cannot be used to reproduce in its entirety the developmental changes in steroidal response to gonadotropins associated with normal puberty.     

Inhibition of ovarian aromatase by prolactin in vivo

Ovarian aromatase activity was inhibited by prolactin treatment in both cycling rats and immature gonadotropin-primed animals. Cycling rats were injected s.c. with prolactin (4 mg/kg BW) beginning on diestrus 1. Aromatase activity in the largest follicles on diestrus 2 and on proestrus was reduced relative to controls. None of the prolactin-treated animals had ovulated when examined on the expected estrus morning. In other experiments, the immature pregnant mare's serum gonadotropin (PMSG)-primed rat was used as a model for the cycling rat. Microsomal aromatase activity but not the activity of the C17-21 lyase was reduced in ovaries of animals injected with prolactin [6 IU (200 micrograms)/rat] 48 h after PMSG administration and sacrificed 4 h later. Furthermore, aromatase activity was significantly increased when endogenous prolactin was inhibited by bromocryptine (CB-154). The effect of LH treatment, on the other hand, was to increase both aromatase and lyase activities. However, prolactin pretreatment did not consistently prevent the LH-induced increase in aromatase activity. The results suggest that inhibition of ovulation by prolactin is mediated, at least in part, by direct inhibition of aromatase activity.     

Increased mortality during early embryonic development after in-vitro fertilization of rat oocytes

Immature female rats (60-65 g) were injected with 4 i.u. PMSG on Day -2, and allocated to 3 groups. For Groups I and II, unmated donors were killed 67-69 h after PMSG injection, shortly after the expected time of ovulation. Oocytes were recovered from the oviducts and transferred immediately into the oviduct of mated recipients (Group I) whose ipsilateral ovary had been exposed by peeling back the bursa, preventing endogenous oocytes from entering the oviduct, or were fertilized in vitro (Group II) and were transferred 16-18 h later. Rats in Group III were allowed to mate and half were killed 6 h after mating. The fertilized oocytes were then incubated for 10-12 h until transfer. The remaining rats in Group III were killed 16-18 h after mating and fertilized oocytes were collected and transferred immediately. Recipient rats were killed on Days 2, 5, 8 and 20. Zygotes resulting from in-vitro fertilization (Group II) were as able as those fertilized in donors (Group III) or recipients (Group I) to develop to the 2-cell stage, but underwent significantly greater embryonic loss beyond this stage of development. There was a slower rate of development of such oocytes to the blastocyst stage (Day 5) and a lower mean weight of implantation sites (Day 8). Transfer of zygotes after in-vitro fertilization resulted in a loss of 35% of the embryos at the time of implantation. These results suggest that in-vitro fertilization of rat oocytes leads to defects in the embryos causing a delay in early embryo development and a large number of implantation losses.     

Rat embryo quality and production efficiency are dependent on gonadotrophin dose in superovulatory treatments

The present study was performed to optimize a superovulation protocol in rats in order to produce a large number of good-quality embryos suitable to develop rat embryonic stem (rES) cells. We first evaluated the ovulation kinetics of three rat strains: Wistar, Fisher and ACI/N. Animals (n=30 per strain) were treated with 50 IU of pregnant mare serum gonadotrophin (PMSG), and ovulation was induced with 50 IU of human chorionic gonadotrophin (hCG) 50 h apart. Next, we evaluated the dose-response curves of PMSG and hCG in Wistar rats in order to obtain the highest number of embryos. The parameters evaluated for superovulation efficiency were: percentage of mated females, percentage of pregnant females and the average number of embryos collected per female. The results of these experiments suggested that the best dose combination was 50 IU for each hormone. Subsequent experiments, again with Wistar rats, were designed to test which of four hormonal combination treatments (30/30, 30/50, 50/30, and 50/50 IU of PMSG/hCG) will produce the largest numbers of good-quality embryos. Embryo quality was evaluated by embryo development uniformity, embryo morphology, embryo survival in an in vitro culture and embryo ability to generate rES-like cells. Results from these experiments showed that 30/50 IU of PMSG/hCG was the treatment that induced the best embryo quality. In conclusion, our results indicated that, in Wistar rats, the most appropriate hormonal combination dose for superovulation protocols with high number of good-quality embryos was 30 IU of PMSG and 50 IU of hCG given 50 h apart. We are performing further studies with rES-like cells produced with the present methodology to evaluate if they are able to participate in the production of germ-line chimeras.     

Immunocytochemical localization of aromatase in immature rat ovaries treated with PMSG and hCG,and in pregnant rat ovaries

Summary Immunocytochemical localization of aromatase cytochrome P-450 was examined in immature rat ovaries treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG), and in pregnant rat ovaries. It is well known that PMSG and hCG treatments induce ovulation about 12 h after hCG injection.At 24 h after hCG injection, many antral follicles were recognized in immature rat ovaries and only the granulosa cells in the antral follicles were stained weakly with the anti-aromatase antibody. At 0 to 9 h after hCG injection, in addition to the antral follicles, some large Graafian follicles could be observed in the rat ovaries, and the granulosa cells of these follicles were positively stained for aromatase. Each follicle was surrounded by the basal lamina which shows lineally distinct positive reaction against anti-laminin antibody. At 12 h after hCG injection, some large Graafian follicles without oocyte were weakly positive to the anti-aromatase antisera, and the outline of their basal lamina stained with anti-laminin antibody became irregular in shape and fragmentous. At 15 to 18 h after hCG injection, the luteinized cysts could be seen, and the granulosa-lutein cells of these cysts were almost negative for aromatase. Fragmentous reaction to the anti-laminin antibody was observed around the luteinized cysts.In the ovaries of day 4 in pregnancy, only the granulosa cells of the large antral follicles were weakly stained, but corpora lutea negatively reacted to the anti-aromatase antibody. At 7 to 19 days in gestation, both the granulosa cells of antral follicles and pregnant luteal cells were positively stained against aromatase antisera. The luteal cells were increased in size during pregnancy. And weakly positive reaction was detected on day 7 of pregnancy, then the immunoreaction became stronger in the corpora lutea on day 15 and 19 of pregnancy.The localization of aromatase was immunocytochemically examined in immature rat ovaries treated with PMSG and hCG injection, and the reaction of the granulosa cells of the antral follicles against anti-aromatase antibody became strongly positive about 12 h before ovulation and the became very weak suddenly after ovulation. In rat-ovaries, the pregnant corpora lutea was positively stained for aromatase after day 7 of pregnancy.This study was supported by Grants from the Ministry of Education, Science and Culture, Japan, and from USPHS Research Grants HD04945, USA     

Effect of dihydrotestosterone on the growth and function of ovarian follicles in intact immature female rats primed with PMSG

Intact, immature female rats were primed with PMSG and treated with 4 injections of DHT. DHT given at 0, 12, 24 and 36 h caused a significant decrease in the ovulation rate 72 h after the PMSG treatment. Concurrent treatment with oestrogen reversed the inhibitory effects of the androgen. The androgen effect was apparently exerted directly on the ovary since DHT did not alter the surge of LH and FSH which occurred at 58 h after PMSG treatment. The DHT inhibition of ovulation was observed in the treatment cycle as well as in subsequent cycles which followed a second PMSG injection. This finding suggests that intermediate size follicles were also adversely affected by the androgen. To confirm that androgen affects follicles of all size ranges, follicles less than 200 microns, 200-400 microns and greater than 400 microns in diameter were isolated from the ovaries of rats treated with PMSG and DHT or the vehicle. The follicles were isolated by density gradient separation of follicles followed by filtration with pre-calibrated Teflon sieves. In some experiments, granulosa cells were also harvested from isolated follicles. DHT treatment did not affect the numbers of follicles of any size but did reduce the oestrogen content of follicles of all sizes. Follicles from DHT-treated animals contained fewer granulosa cells and the cells from treated animals had lower aromatase activity than did cells from control rats. Taken together, these findings suggest that DHT reduces the ovulation rate by decreasing the number of granulosa cells/follicle and by altering the oestrogen synthetic abilities of the cells. All follicles, regardless of size, were sensitive to androgen treatment.     

Superovulation in immature and mature Chinese hamsters

Mature female Chinese hamsters ovulate an average of 8.8 ± 1.0 (mean ± SD) eggs per female in each estrous cycle. Superovulation can be induced in both immature and mature females by subcutaneous or intraperitoneal injections of pregnant mare serum gonadotropin (PMSG) and either human chorionic gonadotropin (hCG) or pituitary luteinizing hormone (PLH). The best superovulation in immature females was induced by the administration of 15 IU of PMSG followed 72 hr later by injection of 15 IU of hCG (about 25 eggs per female) or 0.2 mg (200 IU) PLH (about 46 eggs per female). Ovulation started about 13–15 hr after administration of hCG (or PLH) and was completed during the next 5–6 hr. Superovulation in mature females could be induced by injecting PMSG any day of the estrous cycle, but the best superovulation (about 39 eggs per female) was induced by injecting 15 IU of PMSG on day 1 (day of ovulation) followed by the injection of 0.4 mg of PLH 72 hr later. When immature females treated with the best superovulatory protocol were mated on the evening of PLH injection, only 5% of the eggs were found fertilized 50 hr after PLH administration. On the other hand, about 60% of the eggs were found fertilized in mature females mated following treatment with the best superovulatory protocol. The majority (83–85%) of superovulated eggs obtained from both immature and mature females were normally fertilized in vitro.     

Inhibition of ovulation in the gonadotropin-primed immature rat by exogenous prostaglandin E2

In the past two decades there have been innumerable reports that prostaglandins (PGs) are essential for mammalian ovulation. However, we have recently found that a relatively low dose of 0.03 mg indomethacin (INDO) sc to PMSG/hCG-primed immature Wistar rats can significantly reduce ovarian PG levels without inhibiting the control ovulation rate of 60+ ova/rat (1–3). In view of this information, the present study was an effort to duplicate the earlier reports that PGs can reverse the “inhibitory” effect of INDO on ovulation. In control animals, which received PMSG and hCG only, the ovulation rate was 63.8 ± 4.5 ova/rat. This rate was reduced to 4.1 ± 1.1 ova/rat when the animals were injected with 1.0 mg INDO at 3 h after hCG. In no instance was this inhibition reversed when the animals were treated with 1.0 mg of PGE₂ or PGF_2α, or a combination of both prostanoids in either a single dose at 3 h after hCG, or in 4× doses at 2-h intervals beginning at 3 h after hCG. Furthermore, in animals that did not receive INDO, the ovulation rate in PGE₂-treated animals was reduced to 20.0 ± 6.7 ova/rat, and in animals treated with PGE₂ and PGF_2α (combined) it was reduced to 19.4 ± 6.5 ova/rat. In summary, not only did the PGs fail to reverse the anti-ovulatory effect of INDO, PGE₂ actually suppressed the ovulation rate.     

Role of progesterone in the modulation of the preovulatory surge of gonadotropins and ovulation in the pregnant mare's serum gonadotropin-primed immature rat and the adult rat

The role of progesterone in the regulation of the preovulatory surge in gonadotropins and ovulation was examined in this study by use of a potent antagonist of progesterone, RU 486 (17 beta-hydroxy-11 beta-[4-dimethyl-aminophenyl]-17 alpha- [prop-1-ynyl]estra-4,9-diene-3-one). The immature rat primed with pregnant mare's serum gonadotropin (PMSG) and the cycling adult animal were the models used to verify the role of progesterone. When RU 486 (200 micrograms/rat) was given as a single dose on the morning of proestrus, there was a significant reduction in the preovulatory surge levels of gonadotropins and ovulation in both animal models. Serum progesterone levels in both models at the time of death on the evening of proestrus were unaltered upon treatment with RU 486. RU 486 did not have any effect on gonadotropin levels in immature rats 7 days after castration. These results show that the actin of RU 486 on the preovulatory gonadotropin surge is due to an antagonism of the action of progesterone on the hypothalamic-pituitary axis. Thus, a role for progesterone in modulating the preovulatory surge of gonadotropins and, consequently, ovulation is strongly suggested.     

Effects of hyperstimulation with gonadotrophins and age of females on oocytes and their metaphase II status in rats

The present study was undertaken to evaluate the effects of hyperstimulation and aging on the number and proportion of oocytes in the metaphase II stage in female Wistar rats. It explored the validity of the hypothesis that a combination of hyperstimulation with pregnant mare serum gonadotrophins (PMSG) and age could compromise, to a greater extent, the oocyte quality as indicated by the proportion of ovulated oocytes in the metaphase II stage. Female Wistar rats were stimulated with varying doses of PMSG and human chorionic gonadotrophins (hCG) and the number and proportion of ovulated oocytes in the metaphase II stage were examined and compared between different groups of young adult (8-10 weeks old) and aging (30-32 weeks old) female rats. While spontaneous ovulation occurred in all young adult rats, only 50% of the aging rats did. The ovulation rate in aging rats was increased from 50 to 93% when non-PMSG-stimulated rats were given a dose of 10 IU of hCG at proestrus. The lower number of ovulated oocytes noted, even in those hyperstimulated with high doses of PMSG/hCG, also indicated a reduction in fertility in aging rats. Under the influence of high doses of PMSG, all aging rats ovulated, but as with the young adult rats, a higher dose of hCG was needed to achieve the maximum number of ovulated oocytes from the PMSG-induced expanded pool of preovulatory follicles. However, the average number of ovulated oocytes in aging rats was, nevertheless, still significantly lower than in young adult rats even when approximation of weight was considered. No consistent significant difference in proportion of normal oocytes was noted within groups and between young adult and aging rats. A lower proportion of ovulated oocytes was arrested at the metaphase II stages when rats, whether they were young adult or aging, were hyperstimulated with 40 IU of PMSG. However, this proportion was restored to normal (about 100%) when a higher dose of hCG, which is a signal responsible for initiating oocyte maturation, was used. Results of the present study showed that there appears to be an age-related reduction of sensitivity of the preovulatory follicles to the ovulation induction signal of hCG and thus higher doses of hCG were needed to ovulate the PMSG-induced expanded pool of dominant follicles. In older rats, apart from the obvious depletion of the pool of follicles, the evidence from the present study suggests that some of these older rats do have follicles, but that these were unable to develop to preovulatory follicles, probably because of the absence of sufficiently high levels of gonadotrophins essential for the initiation of folliculogenesis. PMSG-hyperstimulation can affect nuclear maturation; the proportion of ovulated oocytes not arrested at the metaphase II stage was higher. However, the proportion of ovulated oocytes at the metaphase II was restored to normal by increasing the dose of hCG use. Hence, meiotic aberration in rats is not age-dependent but rather dependent on the amplitude of the luteinizing hormone (LH)/hCG surge present. The results from this study nullified the hypothesis that hyperstimulation in combination with aging would lead to a higher proportion of abnormality in ovulated oocytes with respect to their being at inappropriate meiotic stages.     

Apolipoprotein-E messenger RNA in rat ovary is expressed in theca and interstitial cells and presumptive macrophage, but not in granulosa cells.

Apolipoprotein-E (apoE) is a constituent of various lipoproteins and is a ligand for cellular lipoprotein receptors. Unlike most apolipoproteins, apoE is synthesized in peripheral tissues, including those engaged in steroidogenesis. ApoE expression in adrenal cells inhibits cholesterol utilization for steroid synthesis and blocks signal transduction via the protein kinase-A pathway. In cultured ovarian thecal/interstitial cells, exogenous apoE has been shown to inhibit LH-induced androgen synthesis. These findings support a role for apoE as an autocrine or paracrine factor involved in regulating steroidogenesis. In the present study in situ hybridization was used to identify cell types that express apoE mRNA in ovaries from rats with a 4-day estrous cycle, from pregnant rats, from immature rats treated with PMSG to stimulate follicular development, and from PMSG-treated rats that were subsequently administered hCG to stimulate ovulation and luteinization. ApoE mRNA was localized to theca and interstitial cells of follicles in animals at all stages of the estrous cycle as well as in immature rats treated with PMSG. ApoE mRNA was not detected in oocytes, cumulus cells, or granulosa cells. High levels of apoE mRNA also were expressed by localized clusters of presumptive macrophages in atretic follicles and degenerating corpora lutea. This complex pattern of expression may indicate that apoE has multiple functions in the rat ovary. ApoE made by theca and interstitial cells may act locally as an autocrine factor to regulate androgen production. ApoE made in atretic follicles and regressing corpora lutea may serve to facilitate local transport and reutilization of lipid released as these structures degenerate.     

Ovarian response to PMSG treatment in ewes immunized against oestradiol-17 beta

The effects of active immunization against oestradiol-17 beta on the ovarian response to pregnant mare serum gonadotrophin (PMSG) was investigated in Merino ewes. Immunized (79) and control (41) ewes were synchronized with intravaginal sponges, given either 750 or 1500 i.u. PMSG and then mated to rams or inseminated laparoscopically with fresh diluted semen. All control ewes mated naturally exhibited oestrus and 40 out of 41 control ewes ovulated. The ovulation rate was higher in the controls receiving 1500 i.u. PMSG than in those ewes which received 750 i.u. PMSG (10.2 v. 3.3). Immunization against oestradiol-17 beta resulted in antibody titres varying from 100 to more than 100 000 in plasma taken 1-4 days after mating. The ovarian response increased significantly in the lowest titre group (100-1000) in conjunction with stimulation with 1500 i.u. PMSG. In these ewes the ovulation rate increased over controls (16.7 v. 10.2) as did the total ovarian response, which includes follicles greater than 10 mm diameter (22.3 v. 11.1). The total ovarian response was also increased in those ewes given 750 i.u. PMSG which had titres in the 1000-10 000 and 10 000-100 000 range, but this was not accompanied by significant increases in the ovulation rate. In general, the higher titre levels (greater than 1000) were correlated with decreases in the proportion of ewes showing oestrus and ovulating and in the embryo recovery rate. The 1500 i.u. PMSG treatment group with the highest titres (greater than 10 000) also showed a significant drop in the ovulation rate as compared to the 1500 i.u. PMSG controls.     

Deciduomal response in androgenized rats: effect of age and steroid replacement therapy

Deciduomal response was studied in female rats androgenized with a single injection of 1 mg of testosterone propionate at 5 days of age. Endometrial scratching in immature rats (33 days) elicited a better response in androgenized rats (AF) than in controls (NF) following induction of ovulation or steroid replacement therapy. In adult females receiving cervical stimulation at estrus or induction of ovulation, strong deciduomal response was obtained in NF rats and no response was observed in AF rats. In ovariectomized (OVX) rats receiving 2 mg of progesterone (P), the response in AF was only 50% that of NF rats. Addition of 0.1 mg of estradiol (E2) enhanced the decidualization in NF rats but completely abolished that of AF rats. Following ovariectomy and a period of 12-15 days without any exogenous hormone, an E2 priming treatment (0.2 or 0.5 micrograms) for 3 days followed by a replacement therapy (2.0 mg P + 0.1 or 0.15 micrograms E2) allowed good response in NF rats. The response was reduced by 30-35% in AF rats receiving 0.1 micrograms of E2 during the replacement therapy and by 66% in AF rats receiving 0.15 micrograms of E2. These results indicate that in AF rats the reduction of the response is age dependent, the uterus is more sensitive to E2 than is the uterus of NF rats and the growth response is always submaximal.     

Immunocytochemical localization of aromatase in immature rat ovaries treated with PMSG and hCG, and in pregnant rat ovaries

Immunocytochemical localization of aromatase cytochrome P-450 was examined in immature rat ovaries treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG), and in pregnant rat ovaries. It is well known that PMSG and hCG treatments induce ovulation about 12 h after hCG injection. At 24 h after hCG injection, many antral follicles were recognized in immature rat ovaries and only the granulosa cells in the antral follicles were stained weakly with the anti-aromatase antibody. At 0 to 9 h after hCG injection, in addition to the antral follicles, some large Graafian follicles could be observed in the rat ovaries, and the granulosa cells of these follicles were positively stained for aromatase. Each follicle was surrounded by the basal lamina which shows lineally distinct positive reaction against anti-laminin antibody. At 12 h after hCG injection, some large Graafian follicles without oocyte were weakly positive to the anti-aromatase antisera, and the outline of their basal lamina stained with anti-laminin antibody became irregular in shape and fragmentous. At 15 to 18 h after hCG injection, the luteinized cysts could be seen, and the granulosa-lutein cells of these cysts were almost negative for aromatase. Fragmentous reaction to the anti-laminin antibody was observed around the luteinized cysts. In the ovaries of day 4 in pregnancy, only the granulosa cells of the large antral follicles were weakly stained, but corpora lutea negatively reacted to the anti-aromatase antibody. At 7 to 19 days in gestation, both the granulosa cells of antral follicles and pregnant luteal cells were positively stained against aromatase antisera.(ABSTRACT TRUNCATED AT 250 WORDS)