Two distinct placental lactogen-like substances in serum during mid-pregnancy in the rat.

Two forms of placental lactogen (PL), designated PL-I and PL-II, have been reported in the serum of pregnant rats; PL-I was secreted during mid-pregnancy and PL-II was secreted near term. In the present study, we found that two distinct forms with PL-like activity were secreted into the blood during mid-pregnancy. Serum from day-12 pregnant rats was subjected to high-performance liquid chromatography (HPLC) gel-filtration (TSK-G3000SswXL column), and PL activity was assayed by radioreceptor assay using 125I-labeled rat PRL and hepatocyte plasma membrane from pregnant rats. HPLC-gel filtration separated the PL activity into two peaks with mol wt of 55-60 K and 45-50 K, as estimated by reference to the elution volume of standard proteins. We tentatively designated these peaks PL-alpha and PL-beta, respectively. Considering these mol wt, PL-beta seemed to be identical to PL-I. mRNA was extracted from samples of placenta obtained each day from day 8 to day 12 of pregnancy and analyzed by means of a translation system involving micro-injection into Xenopus oocytes. The time of appearance of the mRNAs corresponding to PL-alpha and PL-beta did not correspond and differed according to the day of pregnancy, suggesting that there are individual mRNAs for each PL in rat placenta. Treatment of PL-alpha and PL-beta with peptide: N-glycosidase F completely abolished the binding activity to their receptors. Also since they were sensitive to glycosidases such as endoglycosidase H, endoglycosidase F and neuraminidase, these PLs were considered to possess N-linked glycoresidue(s) and to be sialylated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human chorionic gonadotropin and luteinizing hormone-releasing hormone reverse the blockade of ovulation in pregnant mare's serum gonadotropin-primed immature rats by the anti-androgenic drug, hydroxyflutamide

The present study was designed to examine mechanism(s) of the anti-ovulatory action of the anti-androgen, hydroxyflutamide (OH-F). Prepubertal rats were treated with 4 IU pregnant mare's serum gonadotropin (PMSG) (day -2) to induce first estrus and ovulation. They received OH-F in sesame oil or oil alone at 08:00 and 20:00 h on day 0 (the day of proestrus) and ovulations were assessed on the morning of day 1. Eighty-three percent of control animals ovulated with a mean of 7.7 +/- 1.1 corpora lutea per rat. Hydroxyflutamide blocked ovulation in all but 2 of the 12 rats receiving this drug alone. All of OH-F treated rats that received 5 and 25 IU human chorionic gonadotropin (hCG) ovulated with means +/- SEM of 9.1 +/- 0.1 and 7.3 +/- 1.4 corpora lutea per rat, respectively. The dose of 0.2 IU hCG was essentially ineffective, while the effect of 1.0 IU hCG was intermediate. At the dose of 20 ng and above (100 and 500 ng) luteining hormone-releasing hormone (LHRH) completely overcame the ovulation blockade in the OH-F treated animals, while a 4-ng dose was ineffective. At 18:00 h on the day of proestrus, serum LH levels in control animals were 17.56 +/- 2.60 ng/mL, which were 920% above basal levels (1.90 +/- 0.13) indicating a spontaneous LH surge. This surge was suppressed in OH-F treated rats. Injection of LHRH, at the dose of 20 ng and above, reinstated the LH release in OH-F treated animals. Thus, the anti-androgen, OH-F, inhibits ovulation in PMSG-treated immature rats through its interference with the preovulatory LH surge; the inhibition can be reversed by hCG or LHRH. Hydroxyflutamide does not appear to interfere at the level of the pituitary, but may have direct action at the hypothalamic and (or) extrahypothalamic sites involved in the generation of positive feedback signals that control LH release.     

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     

Rat placental luteotropin: initial secretion and luteolytic quality

The secretion of placental lactogen begins early in pregnancy. Previous studies indicate that rat placental lactogen (rPL) is secreted from Day 8 of pregnancy and that it is luteolytic as well as luteotrophic. This study establishes the onset of both the luteotrophic and the luteolytic effects of placental lactogen in pregnant rats subject to timed hypophysectomy. Pregnancy was preserved in all groups with the administration of dydrogesterone (9 beta, 10 alpha-pregna4,6-diene-3, 20 dione), a progesterone analog, and diethylstilbestrol, an estrogen analog. Plasma progesterone and 20 alpha-hydroxypregn-4-ene-3-one (20-OHP) were measured in serial serum samples by RIA. The data indicate that rPL is secreted as early in pregnancy as the seventh day. Rats hypophysectomized on Day 6 of pregnancy or later had ovaries that contained corpora lutea that secreted increasing quantities of progesterone during pregnancy. On Day 16 serum progesterone values were lowest in animals operated on Days 4 and 5 compared to animals operated on Days 6 or 8. The 20-OHP serum values from animals operated on Days 4 and 5 declined steadily from Day 8 to Day 16. These findings indicate progestational incompetency, which was confirmed morphologically. Thus, rPL secretion begins by Day 7 and it is both luteotrophic and luteolytic.     

Reactivation of regressing corpora lutea by estradiol in the pregnant rat: dependence on placental lactogen

Previous investigations have clearly demonstrated that estradiol maintains corpus luteum function. However, it is unknown whether estradiol can restimulate progesterone synthesis and/or growth of corpora lutea that have already undergone luteolysis. The present study was designed to determine 1) whether estradiol can reactivate the steroidogenic capacity and/or growth of corpora lutea that are deprived of luteotropic support, 2) whether estradiol affects progesterone metabolism, and 3) whether the action of estradiol is related to levels of rat placental lactogen in the peripheral circulation. Rats were hypophysectomized and hysterectomized on Day 12 of pregnancy and were treated between Days 12 and 15 with either estradiol (100 micrograms/day) or 1-cm testosterone implants. Both treatments are known to maintain luteal concentrations of estradiol at physiological levels. In vivo treatment with either estradiol or testosterone prevented the drop in progesterone production and maintained the concentration of serum progesterone at levels found in intact pregnant rats. This action was not due to an alteration in the rate of metabolism of progesterone to 20 alpha-hydroxyprogesterone, since peripheral serum levels and in vitro production of 20 alpha-hydroxyprogesterone were unaffected by estradiol. When testosterone treatment was started 24 and 48 h after hypophysectomy and hysterectomy, at a time when progesterone production had been markedly reduced and luteal growth had ceased, a restimulation of both progesterone synthesis and luteal growth was observed. However, in all cases the ability of estradiol to stimulate progesterone was finite, and corpora lutea ceased to respond by Day 17, coincident with the time that rat placental lactogen became undetectable in the circulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The size of the corpus luteum during pseudopregnancy and pregnancy in the rat.

The corpus luteum in mature Sprague Dawley rats was weighted at the various stages of pseudopregnancy and pregancy. The average size of these corpora lutea was 1.0 +/- 0.10 mg, 1.61 +/- 0.69 mg, 1.90 +/- 0.25 mg, 3.69 +/- 0.36 mg, and 4.37 +/- 0.50 mg on day 2 of diestrus, on days 10-15 of psuedopregnancy, on days 9-10, 14, and 20 of pregnancy, respectively. The fact that the average size of the corpus luteum on days 10-15 of pseudopregnancy was larger than that on day 2 of diestrus is thought to drive from prolonged exposure of the corpus luteum to prolactin. The average size of the corpus luteum on days 9-10 of pregnancy had a tendency to be larger than that on days 10-15 of pseudopregnancy and this seems to demonstrate that the placenta secreted placental lactogen by this stage of pregnancy. The average size of the corpus luteum on day 14 of pregnancy was larger than that on days 9-10 of pregnancy. This phenomenon might be attributed to the presence of large amounts of placental lactogen secreted from the placenta between days 10 and 14 of pregnancy. Furthermore, it was noted that the size of the corpus luteum on day 20 of pregnancy was larger than that of day 14, which suggests that further secretion of placental lactogen continued after day 14 of pregnancy. As there was a remarkable decrease in the number of fetuses on day 20 of pregnancy when overiectomy was performed on day 14 of pregnancy, the ovary was considered indispensable in maintaining pregnancy in the rat.     

Steroidogenic effects of lipoproteins and 25-hydroxycholesterol on luteal and ovarian cells: a comparison of two pseudopregnant rat models

Steroidogenesis was compared between luteal cells from immature pseudopregnant (PSP) rats induced by either 5 IU pregnant mare serum gonadotropin (PMSG) alone or 50 IU PMSG combined with 25 IU human chorionic gonadotropin (hCG). It was also determined whether differences in steroidogenesis existed when the entire ovary (ovarian cells) or just luteal cells from Day 4 PSP rats were exposed in vitro to lipoproteins or 25-hydroxycholesterol (25-OH chol). In the absence of luteinizing hormone (LH), basal steroid accumulation, especially progesterone (P4) was around fourfold greater in luteal cells from rats treated with PMSG alone than from rats receiving PMSG-hCG. However, serum P4 and LH were about fivefold greater in the latter group. It is therefore likely that net cellular cholesterol uptake per luteal cell is lower in the PMSG-hCG treated rats, but this is offset by a much greater mass and number of corpora lutea. Lipoproteins (HDL and LDL) and 25-OH chol stimulated in vitro luteal steroidogenesis from rats treated with PMSG alone or PMSG-hCG, and their responses were virtually identical. Therefore, luteal steroidogenesis in the rat always depends on exogenous cholesterol even though treatment in the preovulatory period with PMS or PMSG-hCG and serum LH and follicle-stimulating hormone (FSH) levels on Day 4 PSP are very different. When ovarian cells from PMSG-hCG treated rats were incubated with LH plus HDL or 25-OHP, the production of 20 alpha-DHP was considerably greater than luteal cell production which may be due to a contribution from nonluteal cells. Indeed, about 30% of the cells in the PMSG-hCG group represent nonluteal components as estimated by weight and deoxyribonucleic acid content.     

Effect of estrogen and placental lactogen on lactogenesis in pregnant rats

The removal of the corpora lutea or ovariectomy on Day 18 of pregnancy induced a rise in serum prolactin 24 h after surgery with a rapid decline to control values 4 h after the surge, only in the ovariectomized group. When hysterectomy was performed in addition to luteectomy or ovariectomy a similar rise in prolactin was obtained. Lactose synthetase activity in mammary tissue was significantly higher in the luteectomized and luteectohysterectomized rats when compared with ovariectomized, ovariohysterectomized rats and the sham-operated group. Estrogen treatment 12 h after ovariectomy increased serum prolactin and lactose synthetase activity to values similar to those measured in luteectomized rats, but this increase was significantly greater when compared with the ovariectomized-nontreated group. Treatment with Tamoxifen did not decrease serum prolactin in the luteectomized rats but lactose synthetase was reduced to values similar to that obtained in ovariectomized rats. Treatment with 2 bromo-alpha-ergocryptine-mesylate (CB-154) prevented the rise in serum prolactin in the ovariectomized, luteectomized and luteectohysterectomized groups, but lactose synthetase activity was lowered to control values (sham-operated rats) only in the luteectohysterectomized rats. According to these findings, rat placental lactogen in the absence of prolactin and progesterone induces lactose synthesis. Estrogen facilitates prolactin but not placental lactogen action on lactose synthetase activity.     

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.     

The use of radioreceptor assay for the detection of pregnancy in the mare

Jugular blood samples were collected between 42-45 days from the last breeding for measurement of pregnant mare serum gonadotropin (PMSG) and progesterone in 46 pregnant mares. A radioreceptor assay (RRA) was developed to measure human chorionic gonadotropin (hCG) and subsequently modified to measure PMSG. Highly purified hCG was iodinated using a lactoperoxidase enzymatic procedure and served as the labeled antigen for both the hCG and PMSG RRA. Standard curves were generated using purified hCG or PMSG. Bovine corpora lutea served as the receptor source. The assay was conducted at 37 degrees C for one hour with a total elapsed time from preparation of the luteal cell homogenate until final results were calculated of 2.5 hours. Twelve of the 46 mares failed to maintain their pregnancy, returned to estrus and reovulated after 45 days post-breeding (non-foaling mares). Thirty-four of the 46 mares delivered foals following a gestation of normal length. Mean concentrations of PMSG in the foaling mares was higher than in non-foaling mares. A concentration of 6.9 I.U. of PMSG/ml was used as the lowest concentration necessary for the confirmation of pregnancy. Five of the mares delivering foals had low concentrations of PMSG and were called non-pregnant. Thus, the incidence of false negatives by RRA was 14.7%. All of the non-foaling mares were correctly diagnosed non-pregnant for an error rate (false positive) of 0.0. Mare Immunological Pregnancy (MIP) tests on the 12 non-foaling mares indicated three false positives - an error rate of 25%. Of the 34 foaling mares, the MIP test indicated 8 inconclusive or false negatives, an error rate of 23.5%. At day 42-45, there was no significant difference in progesterone concentrations (determined by RIA) between foaling and non-foaling mares. RRA is a quick, accurate and quantitative method for measuring PMSG in the mare and can be used to diagnose pregnancy at 42-45 days post-ovulation. Plasma progesterone concentrations at this time were not associated with subsequent pregnancy maintenance as were plasma PMSG concentrations.     

Ovarian response to exogenous hormones in six-week-old lambs.

Crossbred lambs 5--6 weeks old were treated with human chorionic gonadotrophin (hCG) (500 or 1500 i.u.) alone, hCG plus pregnant mare serum gonadotrophin (PMSG) (1000 or 2000 i.u.), 1000 i.u. PMSG alone, or were untreated. PMSG alone and PMSG + hCG increased ovarian weight and uterine weight. PMSG alone stimulated growth and luteinization of follicles but PMSG + hCG induced ovulations and formation of corpora lutea. hCG alone did not change any of the characteristics which were measured. PMSG had a significant effect on the number of vesicular follicles but none of the treatments affected the number of growing follicles.     

Fibrinolytic activity in ovaries of rats and golden hamsters after gonadotrophic stimulation and hypophysectomy

The fibrinolytic activity (FA) in immature ovaries of rats was compared with that of golden hamsters on days 3 and 4 after stimulation with pregnant mares' serum gonadotrophin (PMSG). In addition, FA of immature PMSG-stimulated rats was compared with that of adult rats 18 days after hypophysectomy. By means of the fibrin slide method, FA was localized in serial sections and evaluated semiquantitatively. On PMSG day 3, FA occurred in preovulatory follicles and developing corpora lutea. On PMSG day 4, FA was mostly localized in medullary vessels. This was also the case for hypophysectomized rats. While both species showed a comparably high FA on PMSG day 3, hamsters had a lower FA than rats on PMSG day 4. Similar low FA values were obtained in hypophysectomized rats. Non-vascular FA localization may be attributed to granulosa and thecal cells of preovulatory follicles and to luteinizing cells of developing corpora lutea. Vascular FA localization is ascribed to capillary sprouting in these two structures and to medullary vessels.     

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)     

The effect of pre-ovulatory anaesthesia on ovulation in laparoscopically inseminated domestic cats.

Laparoscopic intrauterine artificial insemination (AI) of electroejaculated spermatozoa was used to compare embryo development and conception rates in domestic cats inseminated either before or after ovulation. Females were given a single (100 iu) injection of pregnant mares' serum gonadotrophin (PMSG) followed by either 75 or 100 iu human chorionic gonadotrophin (hCG) 80 h later. Cats were anaesthetized (injectable ketamine HCl/acepromazine plus gaseous halothane) 25-50 h after administration of hCG for laparoscopic assessment of ovarian activity and for transabdominal AI into the proximal aspect of the uterine lumen. At the time of AI, 23 cats were pre-ovulatory (25-33 h after hCG injection) and 30 were post-ovulatory (31-50 h after hCG injection). Pre-ovulatory females produced 10.5 +/- 1.1 follicles and no corpora lutea compared with 1.9 +/- 0.5 follicles and 7.5 +/- 0.9 corpora lutea for the post-ovulatory group (P < 0.05). Six days later, the ovaries of nine pre-ovulatory and 12 post-ovulatory females were re-examined and the reproductive tracts flushed. On this day, pre-ovulatory cats produced fewer corpora lutea (2.8 +/- 1.5; P < 0.05) and embryos (0.4 +/- 0.3; P < 0.05) than post-ovulatory females (18.9 +/- 3.3 corpora lutea; 4.6 +/- 1.2 embryos). Two of the 14 cats (14.3%) inseminated before ovulation and not flushed became pregnant compared with 9 of 18 cats (50.0%) inseminated after ovulation and up to 41 h after hCG injection (P < 0.05). These results indicate that ovulation in cats is compromised by pre-ovulatory ketamine HCl/acepromazine/halothane or laparoscopy or by both and that electroejaculated spermatozoa deposited by laparoscopy in utero, after ovulation, result in a relatively high incidence of pregnancy. Because ovulation usually occurs 25-27 h after injection of hCG, the lifespan for fertilization of the ovulated ovum appears to be at least 14 h in vivo in cats.     

Effects of aminoglutethimide phosphate on litter size and birth weight in superovulated rats

In this study, litter sizes, birth weights and survival rates of neonates in superovulated rats treated with aminoglutethimide phosphate (AGP) were studied. Young (8- to 9-week old) and adult (14- to 16-week old) rats were treated with 20 or 40 IU PMSG between 1100 and 1300 hours on the day of early diestrus, followed by identical doses of hCG 52 to 54 hours later. These rats received 10 mg AGP on Days 0 to 2 of pregnancy (AGP rats). The duration of pregnancy in the AGP-treated and control rats was about the same. Mean live litter sizes in young and in adult AGP rats treated with 20 IU PMSG and 20 IU hCG were significantly higher (15.5 and 15.4; P<0.01) than in the control rats (11.5 and 12.6). When AGP was given to young and adult rats pretreated with 40 IU PMSG and 40 IU hCG, they produced means of 17.7 and 17.3 live neonates, respectively. The birth weights for young (5.55 to 5.88 g) and adult (5.89 to 5.96 g) AGP rats were smaller than those of respective control rats (6.44 and 6.40 g; P < 0.05-0.01). When the number of neonates was adjusted to 8 per dam for nursing and rearing, all of them were alive at 5 weeks after birth and showed normal growth. However, body weight gains of the progeny of AGP dams were lower than those of controls. These results indicate that rats treated with large doses of gonadotropins and AGP produce larger litter sizes than nontreated controls.     

Changes in progesterone levels in pregnancy block caused by the proximity of a strange male and by the injection of PMSG

Attempts were made to examine progesterone levels during pregnancy block, first initiated by pheromones and then by the injection of pregnant mare serum gonadotropin (PMSG). The proximity of a strange male inhibits the usual increase in progesterone levels, resulting in no activation of the corpora lutea, from the estrous cycle to the pregnancy. Strain differences were observed in the imitation of pregnancy block by the injection of PMSG. The C3H and BALB/c strains required much smaller doses of PMSG for pregnancy block than the C57BL/6 strain did. The changes of progesterone observed in pregnancy block initiated by PMSG were slightly different from that caused by the proximity of the strange male: The progesterone levels temporally increased on the day following the injection of PMSG and then decreased on day 4 in the PMSG-treated group. These results suggest that PMSG imitates the effect of the proximate strange male in the pregnancy block.     

Induction of ovulation, mating, and conception in androgen-treated immature rats

Attempts were made to induce pregnancy in androgen-treated immature rats. Treatment with PMSG alone, which causes ovulation in normal immature rats, failed to cause ovulation in androgenized rats. However, treatment with PMSG plus LHRH was effective in causing ovulation. After ovulation, some of the normal and androgenized rats mated. Normal mated rats became pregnant but androgenized mated rats did not. However, when a pituitary gland was transplanted from a normal rat into the kidney capsule of an androgenized rat to maintain functional corpora lutea, implantation occurred in some of the mated animals. The positive decidual reaction in the uteri of such androgenized rats was similar to that observed in normal rats. These results suggest that the uterine sensitivity to blastocyst implantation of androgenized immature rats may be normal.     

Hormonal regulation of estradiol biosynthesis, aromatase activity, and aromatase mRNA in rat ovarian follicles and corpora lutea

To determine the molecular basis for changes in aromatase (P450arom) activity in rat ovarian follicles and corpora lutea, seven clones for rat P450arom cDNA have been identified and isolated from a rat granulosa cell λgtll cDNA expression library using a 62 mer deoxyoligonucleotide probe (derived from an amino acid sequence of purified human placental aromatase) and a human placental P450arom cDNA probe. One of the rat P450arom cDNA clones contained an insert 1.2 kb in size. Both the human 1.8 kb cDNA and the rat 1.2 kb cDNA probes hybridized to a single species of P450arom mRNA that was 2.6 kb in size. Northern blot analysis revealed that corpora lutea isolated on day 15 of pregnancy contained high amounts of P450arom mRNA, whereas granulosa cells of antral follicles of hormonally primed, hypophysectomized rats (i.e., those from which mRNA was isolated to construct the cDNA library) contained only low amounts of P450arom mRNA. The lower amounts of P450arom in granulosa cells of preovulatory follicles in the estradiol-follicle-stimulating hormone primed hypophysectomized rats were unexpected because follicles incubated in medium containing testosterone substrate produce more estradiol than do corpora lutea isolated on day 15 of pregnancy and incubated under similar conditions. Additional studies will determine the hormonal events responsible for the elevated amounts and constitutive maintenance of P450arom mRNA and aromatase activity in luteal cells in vivo and in vitro.     

Glucocorticoids stimulate the accumulation of lipids in the rat corpus luteum.

We investigated the physiological basis for the trophic effect of glucocorticoids in rat corpora lutea in the absence of pituitary gonadotropins. Immature (Day 29) Sprague-Dawley rats were given eCG and hCG to induce the development of corpora lutea and were hypophysectomized on Day 32. Beginning on Day 40, rats received twice-daily s.c. injections of either dexamethasone (dex; 200 microg/rat/day) or vehicle (controls) and then were killed on Day 44. Plasma 20alpha-dihydroprogesterone, a major steroid produced by the corpora lutea, was higher (p 2-fold of plasma 20alpha-dihydroprogesterone concentration compared to controls. Glucocorticoid receptor protein (about 92 kDa) was detected in both luteal and nonluteal ovarian tissues in this animal model. These effects of glucocorticoids and the presence of the glucocorticoid receptor raise the possibility of a physiological role for glucocorticoids in the rat corpus luteum.     

Effects of progesterone pretreatment on fertility of gilts mated at an induced pubertal estrus

The effects of progesterone (100 mg/d, im) on pubertal fertility were examined in 247 gilts over 3 experiments. In the first experiment, 128 gilts were exposed to progesterone for 0, 2, 4 or 8 d before receiving PMSG (750 IU) 1 d later. The number of large (>4mm) follicles or corpora lutea (CL) were determined on the day of PMSG injection, Day 0 (onset of estrus), Day 1 or Day 10 (n=8). In the second experiment, embryonic survival was observed in 68 gilts after induction of estrus with PG600 (400 IU PMSG, 200 IU hCG). Vehicle or progesterone was previously administered for 2 d to these gilts, and they were allowed 1, 2, or 3 d between the last progesterone injection and PG600. In Experiment 3, a field trial was conducted in which 51 gilts received vehicle or progesterone for 2 d, followed by a 3-d interval before injection of PG600 to induce estrus. The gilts were allowed to farrow. Treatment with progesterone 1 d before PMSG increased (P<0.05) the number and size of preovulatory follicles and increased (P<0.05) the number of corpora lutea. However, the percentage of gilts pregnant by Day 10, the number of embryos recovered per gilt and embryonic survival were reduced (P<0.05) with progesterone pretreatment. Utilizing a smaller dose of PMSG (750 vs 400 IU) with PG600 negated the effects of progesterone pretreatment on ovulation rate. When the interval between progesterone treatment and PG600 was lengthened to 3 d embryonic survival to Day 30 improved but was similar to that of the vehicle/PG600 treated gilts. Fertility, as defined as conception rate and litter size, was similar between gilts exposed to vehicle or progesterone. These results indicate that pretreatment with progesterone up to the day before PMSG might improve follicular development and ovulation rate at the pubertal estrus with a dose of 750 IU of PMSG but not with the 400 IU (PG600). Reducing the dose of PMSG to 400 IU and allowing for 3 d between progesterone and gonadotropin treatment reduced the incidence of uterine infections but resulted in a fertility rate similar to that of gilts receiving PG600 alone.