Apoptotic cell death in the rat adrenal gland: an in vivo and in vitro investigation.

Adrenocortical cell apoptosis was studied by using an established in vivo model, the hypophysectomized rat, and an in vitro model, viz., rat adrenal glands in short-term organ culture. In vivo, apoptosis (biochemical autoradiographic analysis of internucleosomal DNA cleavage) was weak and not apparent until 12-24 h after hypophysectomy. In situ histochemical localization of 3'-end DNA strand breaks revealed that apoptosis in vivo occurred nearly exclusively in subpopulations of zona reticularis cells. Adrenocorticotropic hormone (ACTH) maintenance completely blocked these indices of apoptosis. By contrast, apoptosis (DNA fragmentation) in cultured rat adrenal glands without ACTH was extensive and relatively rapid, being apparent after 1 h and increasing with the duration of incubation. ACTH attenuated (by 44%) but did not completely block apoptosis in vitro. Thus, ACTH appears to be the sole pituitary hormone that forestalls apoptosis of terminally differentiated adrenocortical (zona reticularis) cells. However, the discrepancy between in vitro and in vivo models in terms of the magnitude and rate of DNA fragmentation suggests that, in vivo, other factors finely regulate the magnitude of adrenocortical apoptotic cell death.

Vasoactive intestinal peptide-induced expression of cytochrome P450 cholesterol side-chain cleavage and 17 alpha-hydroxylase enzyme activity in hen granulosa cells.

Experiments were conducted to determine whether vasoactive intestinal peptide (VIP) can regulate expression of cytochrome P450 side-chain cleavage (P450scc) and P450 17 alpha-hydroxylase (P450 17 alpha-OH) mRNA levels and enzyme activity in granulosa cells from nonhierarchal (6-8-mm) follicles. Initial studies demonstrated that immunoreactive VIP is localized within the theca (but not granulosa) layer of both resting (< 0.5-mm follicles) and 6-8-mm follicles, thus providing a potential paracrine mechanism of action for VIP. While short-term (3 h) incubation of granulosa cells with VIP (0.001-1.0 microM) failed to stimulate progesterone production from 6-8-mm follicle granulosa cells, a 4-h culture period in the presence of VIP resulted in increased cyclic AMP (cAMP) accumulation, and a 24-h culture period resulted in progesterone synthesis and increased P450scc mRNA levels; control levels of each endpoint measurement were not altered within the period observed. By contrast, culture with the growth factor transforming growth factor alpha (TGF alpha) in the presence of VIP (1 microM) prevented increases in P450scc mRNA levels and progesterone production. Similar effects of VIP and TGF alpha in the presence of VIP were demonstrated for P450 17 alpha-OH mRNA levels and enzyme activity. Finally, there was an additive effect of VIP (0.1 microM) plus recombinant human (rh) FSH (100 mIU) on the initiation of progesterone production in cultured 6-8-mm follicle granulosa cells compared to the addition of VIP or rhFSH alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical studies of chicken somatotrophs and somatotroph granules before and after hatching.

Immunocytochemical methods were used to gain information about the embryonic development of chicken somatotrophs before and after hatching. To localize growth hormone, anterior pituitary sections were incubated with growth-hormone antibody, and then an indirect peroxidase method was used for light microscopy and an immunogold method for electron microscopy. The earliest evidence of embryonic somatotrophs was seen at 12 days. At this stage somatotrophs were sparse (0.2% of parenchymal cells) and their granules were pleomorphic with elongated ovoid and lozenge shapes predominating. Few of the immunogold-labeled somatotroph granules of the embryo were spherical until 15 days after fertilization. At 18 days, most of the granules were spherical (their shape in the adult chicken). During the six days between the 15-day-old embryo and the 1-day-old chick, the number of gold particles per granule section approximately doubled suggesting an increase in growth hormone content of the granules. This rise was the result of increases in the size of the granule sections and in the concentration of gold particles in the sections. During the embryonic period of 12-20 days, somatotrophs were not more than 3.6% of the anterior pituitary cell population. During the following two days, between the 20-day-old embryo and the 1-day-old chick, the percentage of somatotrophs in the pituitary parenchymal cell population rose rapidly from 3.6% to 20.7% and then increased slowly to 24.6% during the period of 1-5 days after hatching.(ABSTRACT TRUNCATED AT 250 WORDS)

Ovarian innervation develops before initiation of folliculogenesis in the rat.

Sympathetic neurotransmitters have been shown to be present in the ovary of the rat during early postnatal development and to affect steroidogenesis before the ovary becomes responsive to gonadotropins, and before the first primordial follicles are formed. This study was undertaken to determine if development of the ovarian innervation is an event that antedates the initiation of folliculogenesis in the rat, Rattus norvegicus. Serial sections of postnatal ovaries revealed a negligible frequency of follicles 24 h after birth (about 1 primordial follicle per ovary). Twelve hours later there were about 500 follicles per ovary, a number that more than doubled to about 1300 during the subsequent 12 h, indicating that an explosive period of follicular differentiation occurs between the end of postnatal days 1 and 2. Electron microscopy demonstrated that before birth the ovaries are already innervated by fibers containing clear and dense-core vesicles. Immunohistochemistry performed on either fetal (day 19) or newborn (less than 15h after birth) ovaries showed the presence of catecholaminergic nerves, identified by their content of immunoreactive tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. While some of these fibers innervate blood vessels, others are associated with primordial ovarian cells, thereby suggesting their participation in non-vascular functions. Since prefollicular ovaries are insensitive to gonadotropins, the results suggest that the developing ovary becomes subjected to direct neurogenic influences before it acquires responsiveness to gonadotropins.

Immunogold identification of the somatotrophs of domestic fowl of different ages.

The somatotrophs of the pituitary gland of the male domestic fowl were identified by means of an immunoelectron-microscopic method based on gold as the electron-opaque label and an antibody to growth hormone. Gold particles indicating sites of growth hormone were restricted to cells in which virtually all of the granules were labelled. Little, if any, gold label was found outside the granules in these cells designated as somatotrophs, or at sites outside these cells. The size of these gold-labelled secretory granules presumed to contain growth hormone decreased with age, from a mean sectional diameter of 256 +/- 6.2 nm (SEM) at 4-6 weeks to 221 +/- 5.7 nm at 11-18 weeks and 205 +/- 8.6 nm at 24-30 weeks of age. On the basis of these values for mean sectional diameters the change between the first two periods represents a decrease in granule volume of about 36%. However, during the same period the growth hormone concentration of the granules increased. Accordingly, growth hormone content per granule changed little if at all. In contrast, from 11-18 weeks to 24-30 weeks of age there was a decrease of 31% in growth hormone content per granule. These data indicate that growth hormone packaging in the chicken somatotroph changes with age. The first change results in the production of smaller granules of higher growth hormone concentration. During this period growth hormone content per granule remains relatively constant. The later change results in the production of granules of lower growth hormone content than that of younger animals.

Progesterone and testosterone production by dispersed rat placental cells.

Isopycnic separation and unit gravity sedimentation were employed to identify the rat placental cell types capable of producing progesterone and testosterone. Subdivision of Day 12-dispersed placental cells in Percoll gradients revealed that fractions (less than 1.048 g/ml) containing giant cytotrophoblast cells produced greater quantities of progesterone (p less than 0.01) than did fractions (greater than 1.048 g/ml) with equal numbers of placental cells but void of giant cytotrophoblasts. Unit gravity sedimentation of Day 16-dispersed placental cells revealed that when incubated, isolated giant cytotrophoblast cells were capable of producing both progesterone and testosterone. Both of the separation studies strongly suggested that other cell types also produce steroids. However, the biosynthetic capacity of the giant cytotrophoblast cell appeared to be 1000-fold greater than that of the other cell types. Incubation of Day 12-dispersed placental cells with human chorionic gonadotropin or 3',5'-cyclic adenosine monophosphate did not further increase progesterone production as compared to untreated control incubates, suggesting rat placental steroidogenesis is not under trophic hormone control. Electron microscopic observations of giant cytotrophoblast cells revealed a complex ultrastructure suggesting a variety of physiological functions.