J. Th. J. Uilenbroek

Localization of liver-type lipase in rat ovaries and its activity during the estrous cycle and lactation

The conditions for an in vitro assay of liver-type lipase, i.e. an enzyme resembling the lipase releasable from the liver by heparin (liver lipase), in rat ovaries were established. The liver-type lipase activity in the ovaries was almost completely (greater than 95%) located in the corpora lutea and its activity ranged from 0.44 to 0.77 mU per corpus luteum of (pseudo)pregnant rats. Preovulatory ovarian follicles contained very low lipase activity. During the estrous cycle the pattern of lipase activity was similar to that of serum progesterone levels (maximal at diestrus 1 and minimal at diestrus 2). In the individual rats liver-type lipase activity in the ovaries was strongly correlated with serum progesterone and 20 alpha-hydroxyprogesterone. The activity of liver-type lipase also varied during lactation. It was relatively low at an early stage (2-3 days) but increased during later stages of lactation. The serum progesterone level was relatively low in rats lactating for 2-3 or 22-24 days. During the intervening time, its concentrations was elevated. Since serum 20 alpha-hydroxyprogesterone levels varied inversely to progesterone, the total amount of progestagens in blood during lactation remained constant. The cholesterol content of the corpora lutea of the lactating rats was initially high and decreased during the lactation.

Effect of LH on progesterone and oestradiol production in vivo and in vitro by preovulatory rat follicles

Temporal changes in follicular oestradiol production induced in vitro and in vivo by LH were studied. In-vitro changes were measured by incubating preovulatory rat follicles for 12 h, changing the medium every 2 h. Follicles isolated at various intervals after an injection of 10 i.u. hCG were incubated for 2 h to measure changes in oestradiol production in vivo. In both studies there was an increase in oestradiol production lasting 4 h followed by a sharp decline. Progesterone production was also increased by LH in vitro or hCG in vivo, but remained high. A second exposure to LH did not raise oestradiol synthesis, but increased progesterone synthesis in vitro only. The decline in oestradiol production is most probably due to a decrease in C17-20 lyase activity, because addition of testosterone, but not of 17 alpha-hydroxyprogesterone, increased oestradiol production. Incubation of preovulatory follicles in the absence of LH or incubation of follicles derived from animals in which the spontaneous LH surge was blocked by an injection of pentobarbitone sodium also resulted in a decrease of oestradiol and an increase in progesterone production. This oestrogen-progesterone shift was also caused by a decrease in C17-20 lyase activity. The results demonstrate that the changes in steroid production in vivo and in vitro are similar and occur in the presence and absence of LH. It is concluded that the decrease in oestradiol production is dependent on the decrease in the activity of enzymes converting progesterone to aromatizable androgens.

Effect of neonatal undernutrition on serum follicle-stimulating hormone levels and ovarian development in the female rat

1. Female rats were undernourished from birth through replacement for 12 h/d of the lactating mother by a nipple-ligated mother. This resulted in slow growth so that 22 d of age the body-weights equalled those of well-nourished animals at 14 d. 2. At 14, 17, 20 and 22 d groups of eight to ten animals each of undernourished and well-nourished rats were autopsied, serum follicle-stimulating hormone (FSH) concentration was determined by radioimmunoassay, uteruses were weighed and ovaries examined by light microscopy. 3. Undernutrition caused a 3-4 d delay in the third-week drop in serum FSH and in ovarian development as indicated by the numbers of antral follicles and by the maximal diameter of the ovaries and of the largest follicles. 4. It is postulated that the effect of undernutrition on serum FSH may be due to delayed increase in ovarian feedback effectiveness, which may be the result of either the retardation in ovarian development or a continued high level of serum oestrogen-binding protein. It remains uncertain through which mechanisms(s) undernutrition brings about delayed onset of puberty.

Inhibition of induction of 20α-hydroxysteroid dehydrogenase activity in rat corpora lutea in vitro by the progesterone antagonist RU486

To determine if the antiprogestagen RU486 has a direct effect on luteal progesterone secretion, whole corpora lutea or dispersed luteal cells were incubated in the presence of RU486. Whole corpora lutea, isolated from rats at day 5 of pseudopregnancy, were incubated individually in hormone-free medium. The concentrations of progesterone and 20 alpha-dihydroprogesterone in the medium plus corpus luteum was measured before and after 24 h of incubation. In the absence of RU486 the concentration of 20 alpha-dihydro-progesterone increased, while that of progesterone remained unchanged. In the presence of RU486 (230 microM) the concentration of both progesterone and 20 alpha-dihydro-progesterone was increased. Dispersed luteal cells were incubated for 24 h in the presence of various amounts of RU486. In the absence and in the presence of 0.2 and 2.3 microM RU486 a high ratio between 20 alpha-dihydro-progesterone and progesterone was found, while in the presence of 23 microM RU486 the concentrations of progesterone and 20 alpha-dihydro-progesterone were equal. 20 alpha-Hydroxysteroid dehydrogenase (20 alpha-HSD) activity measured in luteal homogenates started to increase between 6 and 12 h of incubation. This increase could be prevented after incubation of the corpora lutea in the presence of 23 or 230 microM RU486 for 24 hrs. It is concluded that the progesterone antagonist RU486 can have a direct effect on luteal progesterone production. RU486 prevents the increase in 20 alpha-HSD activity that normally occurs during in vitro incubation. However, since these effects in vitro can only be obtained with high concentrations of RU486, it is unlikely that this antiluteolytic effect plays a role after injection of RU486 in vivo.

Pituitary Responsiveness to LH-RH in Intact and Ovariectomized Androgen-Sterilized Rats

Serum LH changes in response to LH-RH injection were measured in intact and ovariectomized, steroid-treated female rats which were androgenized neonatally with 1,250 microgram testosterone propionate (TP) on day 5. At a dose of 20 ng LH-RH/100 g b.w., serum LH levels in intact rats increased over pre-injection levels, and at a dose of 100 ng LH-RH/100 g b.w., LH concentrations 15 min after injection were higher in nembutal-blocked proestrous rats than in androgen-sterilized rats. However, the ovulation response was not different between the groups. In ovariectomized estradiol benzoate (EB)-treated, androgen-sterilized rats, serum LH concentrations 15 and 60 min after LH-RH injection were lower than in similarly treated control rats. This effect was not secondary to the anovulatory state of the animal, since it also occurred in ovariectomized EB-treated prepuberal rats and in rats ovariectomized prepuberally and treated with EB in adulthood. Also, after treatment with 5alpha-dihydrotestosterone propionate (5alpha-DHTP), pituitary responsiveness to LH-RH in androgen-sterilized rats was lower than in control rats, which suggests that the subnormal response in the estrogen-treated rats was not due to a relative insensitivity to estrogen in the androgen-sterilized rats. The relatively high pituitary responsiveness to LH-RH in intact androgen-sterilized rats is probably due to the high circulating estrogen levels. The subnormal pituitary responsiveness to LH-RH after ovariectomy and estradiol treatment suggests, in addition to an effect on the hypothalamus, also a direct effect of neonatal androgen administration on the pituitary.

A Possible Direct Effect of Prolactin on Follicular Activity

To examine whether high serum prolactin levels inhibit follicular maturation, prolactin was injected during diestrus of intact female rats or endogenous prolactin levels were raised by applying a suckling stimulus. Injections of rat prolactin (100 micrograms per injection) given 2 and 1 days before proestrus resulted in a lower estradiol production by proestrous follicles during a 4-h incubation period than follicles isolated from control rats. In 4 out of 7 animals this occurred without a change in serum progesterone and luteinizing hormone (LH) concentrations. In the 3 remaining animals corpus luteum function was activated. In these animals serum LH concentrations were decreased and follicular estradiol production was further suppressed. To study follicular development in the presence of suckling-induced hyperprolactinemia, the following experiment was performed. Removal of a 5-pup litter at Day 13 (0900 h) of lactation (Day 1 = day of parturition) resulted in ovulation at Day 16. Replacement of a new litter 24 h after litter removal did not interfere with ovulation on Day 16. This procedure allowed the study of follicular development between Days 14 and 15 in the presence of raised serum prolactin levels. It appeared that this treatment did not affect follicular growth, but in vitro estradiol production by preovulatory follicles isolated at Day 15 was lower than in follicles isolated from nonlactating animals. In 3 out of 13 animals corpus luteum function was reactivated. In these animals LH levels and follicular estradiol production were significantly suppressed. Treatment with bromocriptine (1 mg per injection) on Days 13 and 14, in addition to litter replacement, restored the high estradiol production at Day 15 without affecting serum LH concentrations. The results of this study demonstrate that in the presence of high prolactin levels, follicular estradiol production is low. The inverse relation between prolactin and follicular estradiol production in the presence of unchanged serum LH levels suggests that prolactin can have a direct action on estrogen biosynthesis of follicle cells.