ABSTRACT

To determine whether discernible alterations in neuroendocrine and/or ovarian function precede the loss of regular oestrous cycles in ageing female rats, the present study examined the pattern of gonadotrophin secretion near the time of ovulation and the pattern of ovarian steroid secretion in the early morning of prooestrus in middle-aged (10–12 months old) females displaying regular oestrous cycles and compared these with young (4 months old) animals. In addition, the subsequent reproductive patterns in these animals were observed and correlations between the changes in hormonal profiles and the decline in regular reproductive cyclicity were established. In middle-aged females which subsequently ceased to display regular oestrous cycles (middle-aged non-regular; M-NR) within 1–2 months, the pro-oestrous surge of LH was significantly reduced in magnitude. There was no difference in the LH surge between young females and middle-aged animals which maintained regular oestrous cycles (middle-aged regular; M-R) for at least 2 months. There also was no difference in the magnitude of the pro-oestrous FSH surge or in the secondary rise in FSH in the early morning of oestrus among young, M-R and M-NR females. In a separate group of middle-aged females which subsequently became M-NR, serum concentrations of both oestradiol and testosterone in the early morning of pro-oestrus were markedly raised over those in the young and M-R groups. We suggest that an earlier rise in circulating oestradiol and testosterone concentrations in middle-aged females over many consecutive regular oestrous cycles may gradually render the central nervous system less responsive to the stimulatory feedback effects of ovarian steroids on LH release. As a consequence, the pattern of the pro-oestrous LH surge may be progressively altered in ageing rats until it is inadequate for regular ovulatory function.

J. Endocr. (1984) 100, 43–50

Numerous studies have shown that a variety of cytokines are involved in the regulation of ovarian function. Interleukin-6 (IL-6), has been found in follicular fluid. In this report we show by immunocytochemical methods the localization of the extracellular domain of the IL-6-receptor and its associated signal transducer glycoprotein gp 130 on the surface of granulosa cells (GCs). The possibility that IL-6 may also influence the basal and FSH-stimulated production of estradiol (E2) and progesterone (Prog) by GCs in vitro was also investigated. GCs were obtained from infertile patients undergoing in vitro fertilization and embryo transfer treatment and cultured for 72 h or were given increasing concentrations of human recombinant IL-6 (8--128 pg/ml) in the absence or presence of FSH (96 U/ml). For the time-course studies, FSH-stimulated GCs were treated in the absence or presence of IL-6 (128 pg/ml) and supernatants were assayed at 24 h intervals (24-96 h) for E2 and Prog productions. The results show that increasing amounts of IL-6 significantly inhibit E2 production in the absence or presence of FSH vs untreated controls (P=0.025 at IL-6=128 pg/ml and P=0.016 at IL-6=16 pg/ml respectively). IL-6 also inhibited FSH-stimulated but not unstimulated Prog release (P=0.038 at IL-6=8 pg/ml). These findings suggest that IL-6 may be one of the factors that plays a local regulatory role in the course of FSH-stimulated E2 and Prog release. The time-course studies of the effect of the absence or presence of IL-6 demonstrated a significant inhibitory effect on both E2 and Prog secretion (P<0.001) by FSH-stimulated GCs. As infections of the female reproductive tract are often accompanied by elevated local IL-6 levels, this factor may be one of the links leading to endocrine reproductive dysfunction during genital infections.

SUMMARY

In post-partum lactating rats, sucking by the young was associated with high prolactin release and maintenance of lactation but severe inhibition of LH and FSH release and suspension of oestrous cycles. Shortly after the pups were removed on day 22 post partum, LH and FSH release returned to normal and oestrous cycles resumed. Twice-daily injections of ergocornine methanesulphonate (ERG) into mothers beginning at 5 or 7 days post partum, resulted in sustained inhibition of prolactin release and diminished milk secretion. By frequent exchange of pups between control and ERG-treated mothers, it was possible to maintain vigorous sucking and almost normal pup growth despite low serum prolactin levels and diminished lactation. In these rats, serum levels of LH remained low during 11 or more days of treatment with ERG, but serum FSH was consistently higher than in untreated control mothers. After 11 or more days of ERG treatment, most rats showed a return to normal LH and FSH release and resumption of oestrous cycles. These results suggest (a) that the sucking stimulus rather than high prolactin levels in the circulation is mainly responsible for inhibition of LH and FSH release during the first 11 days post partum, (b) that the sucking stimulus acts to increase prolactin and inhibit LH release by separate hypothalamic mechanisms, and (c) that administration of ERG results in diminished prolactin release and lactation, and in increased release of FSH and subsequently of LH with earlier resumption of oestrous cycles.

Survivin (BIRC5) is a cell survival gene that is overexpressed in endometrial cancer and has been implicated to have a physiological role in normal endometrial function. To determine whether survivin gene expression is regulated by reproductive steroid hormones in the human endometrium, RNA was prepared from normal cycling women in the proliferative and secretory phases of the menstrual cycle. RNA was also isolated from 21 endometrial biopsies from premenopausal women at baseline and following 3 months of treatment with depot medroxyprogesterone acetate. Finally, RNA was isolated from endometrial biopsies from ten healthy postmenopausal women participating in a clinical trial of estrogen replacement therapy at baseline and following 6 months of treatment with conjugated equine estrogen. Quantitative RT-PCR analysis was used to determine survivin, insulin-like growth factor binding protein 1 (IGFBP1), Ki67, and IGF1 gene expression levels. Survivin gene expression was highest in the proliferative phase of the menstrual cycle and showed a statistically significant 4-fold increase in expression following chronic treatment with estrogens; this was strongly correlated with increased Ki67, a marker of proliferation. Survivin gene expression decreased 4.6-fold following chronic progestin treatment in the human endometrium. These data suggest that survivin transcript is regulated by estrogens and progestins in the disease-free human endometrium. The data also suggest that survivin transcript may be used as a biomarker of estrogen and progestin treatment efficacy, but validation studies must be conducted to support this conclusion.

Parathyroid hormone (PTH) stimulates bone resorption as well as bone formation in vivo and in organ culture. The catabolic actions of PTH have been recognized in patients with hyperparathyroidism, or with acute infusion of the N-terminal 1–34 fragment of human PTH (hPTH1–34). Whereas the anabolic actions of daily injection with PTH have been well studied in both humans and mice, the catabolic actions of PTH on murine bone remain to be defined. To do this we sought to create a model with short-term, sustained hyperparathyroidism using osmotic infusion pumps. We treated 10-week-old female C57BL/J6 mice with continuous infusion of hPTH1–34 (8.1 pmol/0.25 μl per h, equivalent to 40 μg/kg per day) or vehicle for 2 weeks, using Alzet osmotic pumps. Bone mineral density (BMD), serum total calcium, hPTH1–34, mouse intact PTH (mPTH1–84), osteocalcin and mouse tartrate-resistant acid phosphatase (mTRAP) activity, and microarchitectural variables of the distal femur were measured. Separately, we compared the effects of intermittent daily injection of hPTH1–34 (40 μg/kg per day) with continuous infusion of hPTH1–34 on BMD and bone markers. Exogenous hPTH1–34 was detected only in the PTH-infused mice. Both intermittent and continuous treatment with hPTH1–34 markedly suppressed endogenous mPTH1–84, but only the latter induced hypercalcemia. Daily PTH injection significantly increased both serum osteocalcin and mTRAP, while continuous PTH infusion showed a strong trend to stimulate mTRAP, with a slight but non-significant increase in osteocalcin. There were significant differences in BMD at all sites between animals treated with the same daily dose of intermittent and continuous hPTH1–34. Microcomputed tomography (μCT) analysis of the distal femurs revealed that hPTH1–34 infusion significantly decreased trabecular connectivity density (P<0.05). Thus, the murine bone response to continuous PTH infusion was quite different from that seen with daily PTH injection. Short-term infusion of hPTH1–34 appears to be a good model to study the mechanisms underlying the catabolic action of PTH in mice.

Excessive fat accumulation in liver (hepatic steatosis) predisposes to hepatic functional and structural impairment and overall metabolic risk. Previous studies noted an association between hepatic steatosis and age in humans and rodents. However, the mechanisms leading to age-associated hepatic fat accumulation remain unknown. Earlier work from our group showed that β-adrenergic receptor (β-AR) levels and β-AR-stimulated adenylyl cyclase activity increase in rat liver during aging. Here we investigated whether age-associated increases in β-AR signaling play a role in augmenting hepatic lipid accumulation. We demonstrate an increase in hepatic lipid content during senescence and a significant correlation between hepatic fat content and stimulation of adenylyl cyclase activity by the β-AR agonist isoproterenol in rat liver. Isoproterenol administration to young and old rodents in vivo increased hepatic lipid accumulation. Furthermore, in vitro overexpression of β₁- and β₂-AR subtypes in hepatocytes from young rodents increased cellular lipid content, whereas inhibition of β-ARs by receptor subtype-specific inhibitors reduced lipid levels in hepatocytes from senescent animals. Isoproterenol-induced hepatic lipid accumulation in vivo was prevented by the β-AR nonselective blocker propranolol, suggesting a novel therapeutic effect of this class of drugs in hepatic steatosis. Acipimox, which inhibits adipose tissue lipolysis, did not alter isoproterenol-mediated hepatic fat accumulation; thus β-AR responsive hepatic lipid accumulation does not appear to be related primarily to altered lipolysis. These findings suggest that augmented hepatic β-AR signaling during aging may increase lipid accumulation in liver and advocate a possible role for β-adrenergic blockers in preventing or retarding the development of hepatic steatosis.