Noboru Takasugi

Cytological basis for permanent vaginal changes in mice treated neonatally with steroid hormones.

Publisher Summary Since the vaginal-smear method for determining the stages of the estrous cycle was established in rats by Long and Evans, this technique has become indispensable for investigations of the hypothalamo-hypophysio-ovarian system in rats and mice. The vaginal epithelium responds to estrogen, progestin, or androgen, showing either proliferation and cornification or mucification. Fully cornified smears indicate the estrous state caused by estrogen from endogenous or exogenous sources. The epithelium reversibly responds to estrogen and becomes atrophic in its absence. Thus, proliferation and cornification of the vaginal epithelium have long been considered a reliable criterion of the estrous state caused by estrogen in laboratory rodents. In 1936, Pfeiffer demonstrated that transplantation of testes of newborn male rats into littermate females induces a persistent-estrus state when they became adults. Neither ovulation nor luteinization takes place in the ovaries of these animals. A similar persistent-estrus state is also induced by neonatal administration of androgen or estrogen to rats and mice; this state is called the “early androgen or estrogen syndrome.” Vaginal cornification continues in persistent-estrus animals but ceases following ovariectomy. Accordingly, persistent vaginal cornification is an ovary-dependent (estrogen-dependent) phenomenon because of permanent changes in sexual differentiation of the hypothalamus. However, ovary-independent (estrogen-independent) persistent vaginal cornification has recently been demonstrated in rats and mice treated neonatally with estrogen or androgen. In this chapter, the cytological basis for the development of estrogen-independent vaginal proliferation and cornification is discussed, with attention to its implication in tumorigenesis.

MITOTIC ACTIVITY OF VAGINAL EPITHELIAL CELLS FOLLOWING NEONATAL INJECTIONS OF DIFFERENT DOSES OF ESTROGEN IN MICE

In C57Black/Tw mice given injections of 1 μg estradiol‐17β (E) for 5 days beginning on the day of birth, and killed a few days after the treatment, the vaginal epithelium showed estrogen‐dependent proliferation and parakeratosis. In contrast, in the mice treated neonatally with 30 μg E for 5 days, the vaginal epithelium exhibited estrogen‐independent proliferation and cornification or parakeratosis. Two peaks occurred in the mitotic rate in vaginal epithelial cells in the proximal and middle vaginae of the 1 μgE‐treated mice, at 1 and 5 days of age, respectively, while the first peak was lacking in the distal vagina. The mitotic activity in 1 μgE‐treated mice declined to the control level at 60 days. In the 30 μgE‐treated animals also, 2 peaks were found in the mitotic rate at 1 and 7 days in both the proximal and middle vaginae. In contrast to the 1 μgE‐treated mice, although the rate dropped once at 10 days, it increased again at 20 days and remained high thereafter. The second peak at 7 days of age coincided with the active proliferation of nodules appearing in the 30 μgE‐treated mice. In the distal vagina, a peak occurred in the mitotic rate at 7 days without a preceding peak like that observed in the other parts of the vagina following the first injection of E on the day of birth.

DEVELOPMENT OF VAGINAL EPITHELIUM SHOWING IRREVERSIBLE PROLIFERATION AND CORNIFICATION IN NEONATALLY ESTROGENIZED MICE: AN ELECTRON MICROSCOPE STUDY *

Irreversible proliferation and cornification of the mouse vaginal epithelium were induced by 10 daily injections of 20μg estradiol‐17β starting on the day of birth. Development of the irreversible vaginal epithelium during the period of estrogen injections in early postnatal life was observed under light and electron microscopes. Small electron‐dense cells (A‐cells) in clusters were present in the columnar vaginal epithelium of newborn mice. A‐cells were proliferated by 2 daily estrogen injections. At the sites of A‐cell clumps, large electron‐dense cells (B‐cells) characterized by long winding cytoplasmic processes appeared in mice given 3 daily injections, forming nodules which then fused together to form a layer under the columnar epithelium after 4 daily injections. In mice given 7 daily injections, the primary epithelium was shed by the superficial cornification of the newly formed layer. The B‐cell membrane bore fewer desmosomes than in the basal and intermediary cells of the vaginal epithelium of ovariectomized ‘normal’ adult mice after 5 daily injections of 100μg estradiol‐17β. Hyperplastic epithelial downgrowths in old ovariectomized mice given neonatal estrogen injections contained another type of cells with reduced density which formed much fewer processes and only a few desmosomes (C‐cells).