Stefan Cajander

Ovulation and the mechanism of follicle rupture

SummaryOvarian follicles of twenty-three oestrous rabbits from accurately determined times before and after follicle rupture were studied by light microscopy. Epon embedded material was used, in addition to paraffin sections, to obtain more detailed information. Eighteen animals were given an ovulatory dose of human chorionic gonadotrophin (HCG); ovulation occurs fairly regularly 10–12 hr after HCG.At 4 hr after HCG a prominent oedema appeared in the theca interna region and at 10 hr and later there was an obvious oedema in all layers between membrana granulosa and the surface epithelium; the connective tissue cells were apparently disrupted and widely separated. Already at 4 hr the granulosa cells were slightly dissociated and more so at 8 hr and later.Most distinct changes were found in the germinal epithelium prior to ovulation. The surface cells of the preovulatory follicles increased in size, and from 4 hr and onwards they showed an increasing number of dense and large intracytoplasmic bodies. Such intracellular alterations in the germinal epithelium have not been described before. It is suggested that they may be causally related to follicle rupture.

Steroid-induced polycystic ovaries in rats: effect of electro-acupuncture on concentrations of endothelin-1 and nerve growth factor (NGF), and expression of NGF mRNA in the ovaries, the adrenal glands, and the central nervous system

Previous studies on the effect of repeated electro-acupuncture (EA) treatments in rats with steriod-induced polycystic ovaries (PCO), EA has been shown to modulate nerve growth factor (NGF) concentration in the ovaries as well as corticotropin releasing factor (CRF) in the median eminence (ME). In the present study we tested the hypothesis that repeated EA treatments modulates sympathetic nerve activity in rats with PCO. This was done by analysing endothelin-1 (ET-1), a potent vasoconstrictor involved in ovarian functions, as well as NGF and NGF mRNA expression involved in the pathophysiological process underlying steroid-induced PCO.The main result in the present study was that concentrations of ET-1 in the ovaries were significantly lower in the PCO group receiving EA compared with the healthy control group (p < 0.05). In the hypothalamus, however, ET-1 concentrations were found to be significantly higher in the PCO group receiving EA than in the healthy control group (p < 0.05). Concentrations of ovarian NGF protein were significantly higher in the PCO control group compared with the healthy control group (p < 0.001), and these concentrations decreased significantly after repeated EA treatments compared with those in the PCO control group (p < 0.05) and were found to be the same as those in the healthy control group. In conclusion, these results indicate that EA modulates the neuroendocrinological state of the ovaries, most likely by modulating the sympathetic nerve activity in the ovaries, which may be a factor in the maintenance of steroid-induced PCO.

Acupuncture and exercise restore adipose tissue expression of sympathetic markers and improve ovarian morphology in rats with dihydrotestosterone-induced PCOS

Altered activity of the sympathetic nervous system, which innervates adipose and ovarian tissue, may play a role in polycystic ovary syndrome (PCOS). We hypothesize that electro-acupuncture (EA) and physical exercise reduce sympathetic activity by stimulating ergoreceptors and somatic afferent pathways in muscles. Here we investigated the effects of low-frequency EA and physical exercise on mRNA expression of sympathetic markers in adipose tissue and on ovarian morphology in female rats that received dihydrotestosterone (DHT) continuously, starting before puberty, to induce PCOS. At age 11 wk, rats with DHT-induced PCOS were randomly divided into three groups: PCOS, PCOS plus EA, and PCOS plus exercise. The latter two groups received 2-Hz EA (evoking muscle twitches) three times/week or had free access to a running wheel for 4-5 wk. In mesenteric adipose tissue, expression of beta(3)-adrenergic receptor (ADRB3), nerve growth factor (NGF), and neuropeptide Y (NPY) mRNA was higher in untreated PCOS rats than in controls. Low-frequency EA and exercise downregulated mRNA expression of NGF and NPY, and EA also downregulated expression of ADRB3, compared with untreated rats with DHT-induced PCOS. EA and exercise improved ovarian morphology, as reflected in a higher proportion of healthy antral follicles and a thinner theca interna cell layer than in untreated PCOS rats. These findings support the theory that increased sympathetic activity contributes to the development and maintenance of PCOS and that the effects of EA and exercise may be mediated by modulation of sympathetic outflow to the adipose tissue and ovaries.

Telomerase activity in relation to p53 status and clinico‐pathological parameters in breast cancer

Cell cycle deregulation can occur at different levels in cancer. In human breast cancer it includes overexpression of cyclins D1 and E, down‐regulation of cyclin‐dependent kinase inhibitors and inactivation of the retinoblastoma and p53 tumor suppressor proteins. Telomerase activity is strongly associated with an immortal phenotype and expression of telomerase is linked to the cell cycle. We have recently demonstrated a connection between specific cell cycle defects within the pRB pathway and levels of telomerase activity in breast cancer. In the present study, 106 tumors were investigated for p53 gene and protein status. By single strand conformation polymorphism (SSCP) analysis, 15% showed mutations within exons 5–8 and by immunohistochemistry (IHC), 29% were p53 positive. Tumors with a telomerase activity above median (i.e., telomerasehigh) were significantly associated with p53 protein accumulation (p = 0.004), but not with p53 gene mutations. The strongest telomerase expression was found in tumors with p53 protein accumulation. Morphologic grade, estrogen and progesterone receptor expression differed significantly between the telomerasehigh and telomeraselow groups (p < 0.0001, p = 0.016 and p = 0.046, respectively), but no difference was observed for stage or nodal status. Telomerasehigh tumors were significantly associated with a poor prognosis for node‐negative (N0) patients (p = 0.008), but not for node‐positive (N+) patients, whereas the opposite was demonstrated for tumors with p53 accumulation. The survival data indicated that telomerase expression has biological importance particularly for N0 tumors, suggesting that telomeraselow tumors constitute a group of “pre‐immortalized” tumors with a good prognosis. Int. J. Cancer (Pred. Oncol.) 79:343–348, 1998.

Morphometric characteristics of the vaginal epithelium during the menstrual cycle.

Vaginal biopsies were obtained in 5 healthy women every third day during the menstrual cycle. The specimens were examined with morphometric technique and the mean nuclear size was correlated to values of estradiol and progesterone and caryopycnotic index. Cyclical changes of the histological appearance of the epithelium could be demonstrated. During estrogen influence a proliferation and maturation of the vaginal mucosa was seen whereas addition of progesterone was associated with a shedding of the superficial cell layers and a steady state of the basal part of the epithelium. At ovulation the epithelium reached maximal thickness and maturity. The histological architecture at this time may resemble a human-papilloma-virus (HPV)-infected mucosa. The difficulties in distinguishing an HPV-infected epithelium from normal are discussed.

Cyclin E expression and proliferation in breast cancer

Cyclin E is a part of the cell cycle machinery and aberrantly expressed in several malignancies including breast cancer. Since cyclin E is cell cycle specifically expressed, we wanted to examine the relation between proliferation and expression of cyclin E with special attention to tumours with overexpression of the protein. Seventy-four breast tumours were analysed for the expression of cyclin E by immunohistochemistry and Western blotting and related to the growth fraction determined by Ki-67. Significant correlations were obtained between the growth fraction, the percentage of cyclin E positive cells, the intensity of cyclin E and total amount of cyclin E determined by Western blotting. The majority of the tumours had less cyclin E than Ki-67 positive cells indicating a conserved cell cycle specific expression of the protein which further was supported by flow cytometric analysis of breast cancer cell lines. The cell cycle specificity of cyclin E was found even in tumours with inactivated retinoblastoma protein (pRB) demonstrating the existence of a pRB independent regulation of cyclin E. A fraction of the tumours had considerably elevated cyclin E levels that were not in relation to the proliferative activity as observed for the other tumours. These tumours were in general highly proliferative and considered to overexpress cyclin E. Patients with tumours of high proliferative activity, high total cyclin E levels or disproportionally elevated cyclin E expressions in relation to proliferation had significantly increased risk of death in breast cancer, whereas the intensity of the immunohistochemical cyclin E staining did not affect the survival.

Fine structural demonstration of acid phosphatase in rabbit germinal epithelium prior to induced ovulation.

SummaryThe germinal or surface epithelium covering rabbit Graafian follicles contains occasional small, dark, lysosome-like bodies. After an ovulatory dose of human chorionic gonadotropin (HCG) such bodies gradually increase in size and number. At 8 hr after HCG there is a maximal accumulation in the apical follicle cells; then the dense bodies decrease and just prior to ovulation, 9.5 hr after HCG, only few of them remain in the attenuated surface epithelium.Most of the growing membrane-surrounded bodies probably represent lysosomes, since electron microscopy combined with cytochemistry revealed that many of them contain the lysosomal “marker” enzyme, acid phosphatase.The role of sex steroids and prostaglandins regarding lysosomal growth and labilization is discussed.The close temporal relation between disappearance of the apical surface epithelial lysosomes and disintegration of the underlying tunica albuginea gives further support to our working hypothesis that at least part of the “ovulatory enzymes” emanate from the surface epithelium.The technical assistance of Miss Ingalis Fransson, Miss Kerstin Nilsson and Mrs. Ulla-Britt Westman is greatly appreciated.

Intratumoral variations in DNA ploidy and s‐phase fraction in human breast cancer

To study intratumoral DNA ploidy heterogeneity and S‐phase fraction (SPF) variability, we prospectively collected five different samples from 48 breast carcinomas and each sample was analysed separately by flow cytometry. Aneuploidy rate was 89.6% after analysis of four or five samples. DNA ploidy heterogeneity, i.e., different samples classified as either DNA euploid or DNA aneuploid in the same tumor was seen in 17%, and DNA index heterogeneity, i.e., tumor populations with different DNA indices (DIs) seen in different samples was 44%. A statistical model defining SPF heterogeneity is proposed. SPF heterogeneity as defined by us was 71%, and as expected the SPF heterogeneity rate increased significantly with increasing number of analysed samples. Four or more samples are needed to detect the most deviant (highest) SPF values. An unrecognized intratumor heterogeneity of DNA ploidy and SPF may partly explain the conflicting results reported in the literature on the above prognostic indicators.

Further studies of the surface epithelium covering preovulatory rabbit follicles with special reference to lysosomal alterations

SummaryThe fine structure of the surface epithelium over preovulatory rabbit follicles was examined parallel with visualization of acid phosphatase at the electron microscopical level. Small enzyme positive vesicles were pinched off from the Golgi cisternae and similar vesicles fused and got incorporated into larger lysosomes of dense body type. Some lysosomes appeared in direct continuity with tubular enzyme positive structures. Other possible ways of increase of the lysosomal pool are indicated and discussed.As in previous studies a maximal accumulation of lysosomes was found in the apical epithelium at 8 h after an ovulatory dose of human chorionic gonadotrophin (HCG); thereafter a gradual loss of lysosomes ensued. Before the lysosomes disappeared from the surface epithelium they changed in character. They became more electron-lucent and revealed a fine-fibrillar matrix. Dense bodies deep in the cell interior appeared to communicate with each other and with the extracellular space below the surface epithelium. Openings were never seen towards the peritoneal cavity. The loss of lysosomal content from the apical surface epithelium before follicle rupture appeared in many respects similar to the histamine release process in mast cells.The findings support our working hypothesis that the surface epithelium over Graafian follicles is an essential source of proteolytic enzymes and that these may be released extracellularly and actively contribute to the dissolution of the follicular apex before rupture.

Structural alterations of rabbit ovarian follicles after mating with special reference to the overlying surface epithelium

SummaryRabbit ovarian preovulatory follicles and in particular the overlying surface epithelium were studied by morphological and ultrahistochemical means at different times after mating.By light microscopy an increase of cytoplasmic granules was found in the surface epithelium at the follicle apex 4 h after mating. The granules increased in amount and showed maximal accumulation 8–9 h after mating. They then disappeared at the same time as the connective tissue elements in the underlying tunica albuginea and theca externa disintegrated.Transmission electron microscopy showed that the membrane-bounded granules or dense bodies fused with one another and by 8 h after mating they often changed character and appeared more electron lucent. Furthermore, open communications were found between altered granules and vacuoles and between vacuoles and the extracellular space below the epithelium. Acid phosphatase reaction product was localized to the granules and Golgi cisternae. Not all the dense bodies were enzyme positive. At later stages, close to the time of follicle rupture, the epithelial cells were attenuated and thin, with only a few granules.By scanning electron microscopy it was found that the epithelial cells at the follicle apex increased in size approaching the time of follicle rupture and that their microvilli decreased in number and in size. At 8 h and later, the contours of intracellular granules could be visualized.The results of this study were similar to those found when rabbits were induced to ovulate by HCG-stimulation. This further strengthens the hypothesis that the surface epithelium contributes proteolytic enzymes which help to disintegrate the follicle apex prior to rupture.