Louis V. DePaolo

Follistatin and Activin: A Potential Intrinsic Regulatory System within Diverse Tissues

Until the 1920s, it was assumed that the sole gonadal hormones modulating anterior pituitary glandular secretions were lipid-soluble entities called steroids. Experimental evidence then surfaced during the ensuing decade that demonstrated that the testis also contained a hydrophilic material that might also influence pituitary secretory activity (1, 2). The name “inhibin” was given to this novel substance in view of its ability to inhibit hypertrophy of pituitary cells in castrated rats (2). Work in later years evoked the concept that inhibin could be an important regulator of follicle-stimulating hormone (FSH) secretion (3, 4). Thus, as a consequence of these landmark studies, investigators began a search to elucidate the chemical nature of the inhibins. Although inhibin was discovered as a potential secretory product of the testis, it was almost 45 years later that a nonsteroidal FSH suppressor was demonstrated in the ovarian follicular fluid of cows (5). Ironically, it was the ovarian follicular fluid from which chemists eventually isolated what was most likely the putative inhibin molecule described in the 1920s and 30s (6–9). Chemical characterization of the purified FSH-suppressing material revealed that inhibin is a 31-to 32-kDa heterodimer consisting of two distinct polypeptide chains, α and β. In pig follicular fluid, two inhibin heterodimers, A and B, were isolated and found to consist of a common α-subunit chain linked by disulphide bridges to a βA- or βB-subunit chain, respectively. Both inhibins were essentially equipotent in suppressing FSH secretion in the dispersed anterior pituitary cell bioassay system (6). At long last, the elusive inhibin had been proven a true chemical entity—a major breakthrough in reproductive endocrinology. However, little did anyone know that the gonads produced other FSH-regulatory polypeptide factors besides inhibin, and that the ability of these other factors to modulate FSH secretion could be only the tip of the iceberg with regard to the multi-functional properties of these factors, thereby extending their importance to other areas of biology.

Inhibins, activins, and follistatins: the saga continues.

In 1991, I co-authored a minireview which appeared in this journal and dealt with two protein factors that had recently been isolated from ovarian follicular fluid (1). The two proteins, activin and follistatin, were discovered in the context of their effects on the reproductive system to stimulate and inhibit, respectively, the secretion of follicle-stimulating hormone (FSH) from the anterior pituitary gland (2–5).

Interactions of Δ9-Tetrahydrocannabinol (THC) with Hypothalamic Neurotransmitters Controlling Luteinizing Hormone and Prolactin Release

The effects of delta 9-tetrahydrocannabinol (THC) on hypothalamic norepinephrine (NE) and dopamine (DA) turnover and hypothalamic serotonin (5-HT), 5-hydroxyindole acetic acid (5-HIAA) and LHRH content preceding and during a progesterone- (P) induced LH and prolactin (PRL) surge were investigated in ovariectomized estrogen-primed rats. THC had no effect on basal LH levels, but it inhibited basal PRL levels and blocked the surges of both LH and PRL. The turnover of NE, as estimated by measuring NE depletion after inhibition of tyrosine hydroxylase with alpha-methyl tyrosine (250 mg/kg), in both the anterior (AH) and medial basal hypothalamus (MBH) was significantly inhibited by THC. THC did not significantly affect AH or MBH DA or 5-HT content nor MBH-DA-turnover. Hypothalamic LHRH levels were significantly elevated 4 h after THC administration as compared to the vehicle-injected controls, but pituitary response to exogenous LHRH was not affected. These data suggest that THC inhibits the steroid-induced positive feedback release of LH by reducing NE metabolism and the release of hypothalamic LHRH. Although the mechanism for the inhibition of PRL release by THC is not clear from these experiments, it does not appear that alterations in DA turnover are a contributing factor.

Circulating levels of follistatin from puberty to menopause.

OBJECTIVE To determine the changes in circulating levels of follistatin, a binding protein for activin and inhibin, through the reproductive life cycle in women. DESIGN An open, prospective descriptive study. SETTING An academic endocrine research unit. PATIENTS Prepubertal (n = 10), midpubertal (n = 7), and postpubertal (n = 25) (early adolescent) girls, normal cycling adult women (n = 8), postmenopausal women (n = 17), and men (n = 13) were studied. INTERVENTIONS Normal cycling women were given Nal-Glu GnRH antagonist for 3 days in the follicular phase of the cycle. MAIN OUTCOME MEASURE Serum concentrations of follistatin determined in a heterologous RIA. RESULTS Mean follistatin levels did not change during puberty but were higher in adult and postmenopausal women. Levels of immunoreactive follistatin in men were lower than levels found in normal cycling women and postmenopausal women. Daily immunoreactive follistatin levels during the menstrual cycle remained constant and did not change significantly after ovarian suppression with GnRH antagonist. CONCLUSION Because dynamic changes of serum immunoreactive follistatin do not occur during ovarian activation (puberty), suppression, and age-related ovarian failure, the increase in immunoreactive follistatin levels in adult and postmenopausal women may implicate sources of follistatin other than the ovary.

Age-associated increases in serum follicle-stimulating hormone levels on estrus are accompanied by a reduction in the ovarian secretion of inhibin

In light of recent data demonstrating age-related alterations in the secretion and production of follicle-stimulating hormone (FSH) during the secondary FSH surge on estrus, the following study was conducted to determine the effects of age on periovulatory inhibin secretion. Ovarian venous blood was collected from groups of ether-anesthetized 3- and 7-month-old rats exhibiting 4-day estrous cycles at the following times: 1200 and 2400 h on proestrus and 1600 h on estrus. Following a 10-min collection period, a terminal blood sample was obtained from the abdominal aorta. Peripheral serum concentrations of luteinizing hormone (LH), FSH, estradiol-17 beta (E2), progesterone (P) and testosterone (T) were measured by RIA. Inhibin activity in ovarian venous serum (OVS) was assessed by the ability of OVS to suppress basal FSH secretion from dispersed pituitary cells during a 24-hour culture period. At 1200 h on proestrus, serum FSH (and LH) levels were higher in 7-month-old rats than in younger rats while the FSH-suppressing activity of OVS did not differ between age groups at this time. Bioassayable inhibin activity substantially declined between 1200 and 2400 h on proestrus in both groups. By 1600 h on estrus, serum FSH levels and inhibin secretion were higher and lower, respectively, in the older age group compared to 3-month-old rats. Significant increases in inhibin secretion between 2400 h on proestrus and 1600 h on estrus were observed only in younger rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Examination of a Possible Mechanism by which Anisomycin Suppresses Pulsatile Luteinizing Hormone Release in Ovariectomized Rats

An initial study was performed to ascertain the effects of anisomycin, a reversible inhibitor of protein synthesis, on pulsatile luteinizing hormone (LH) release in adult, ovarietomized (OVX) rats. For this experiment, rats OVX 3-4 weeks earlier were fitted with indwelling atrial cannulae. On the next day (approximately 13.00 h), the rats received a subcutaneous injection of either 100 mg/kg body weight (BW) anisomycin or its saline vehicle. Administration of anisomycin significantly suppressed mean plasma LH levels, mean trough values, and both LH pulse frequency (saline: 6.3 pulses/3 h vs. anisomycin: 2.7 pulses/3 h) and amplitude. To determine whether anisomycin affected anterior pituitary LH responses to LH-releasing hormone (LHRH), a second experiment was performed in which saline- and anisomycin-treated OVX rats were given an intravenous injection of 10 ng/100 g BW LHRH 1.5 h later (14.30 h). Rats then were sacrificed and the anterior pituitary and brain removed. Whereas preinjection plasma LH levels were significantly lower in anisomycin-treated rats, they were significantly higher in anisomycin-treated rats 20 min after LHRH. Consequently, mean maximal increments and percent increments were significantly higher in anisomycin-treated rats. AP LH content and content of LHRH in the medial preoptic and suprachiasmatic nuclei were not influenced by anisomycin treatment. However, median eminence (ME) LHRH concentrations in anisomycin-treated rats were almost double the LHRH levels measured in control rats. A third study was conducted to assess the effects of anisomycin on basal and potassium (K+)-stimulated LHRH release from superfused ME explants.(ABSTRACT TRUNCATED AT 250 WORDS)

Increases in the basal secretion rate of follicle-stimulating hormone (FSH) accompany age-associated changes in serum FSH levels on estrus

Abstract This laboratory has recently reported that by 5–6 months of age, alterations in the secretion and production of follicle-stimulating hormone (FSH) occur in virgin female rats which precedes the age-related disruption of estrous cycles and attenuation of preovulatory gonadotropin surges. Specifically, circulating immunoreactive FSH levels are higher on estrus in rats 5 months and older compared to levels measured in 2- to 3-month-old rats. Therefore, the present study was conducted to explore a possible mechanism for this age-related increase in FSH levels. At 1400 hr on proestrus, estrus and diestrus-1, groups (n = 6–12 rats/group) of 3-and 7-month-old, cyclic rats were decapitated, trunk blood was collected, and anterior pituitary glands were bisected and placed in incubation flasks containing 1 ml media (medium 199). Following a 30-min preincubation period, hemipituitary fragments were incubated for an additional 2 hr. Media and serum FSH levels were quantified by RIA. Levels of FSH were twofold higher in the serum of 7-month-old rats than 3-month-old rats on estrus. Similarly, the basal secretion rate (BSR) of FSH (expressed as ng FSH/mI/2 hr) was significantly (P < 0.05) higher from incubated hemipituitary fragments of 7-month-old estrous rats than from fragments obtained from younger estrous rats (7 month: 1637 ng/ml/2 hr vs 3 months: 1253 ng/ml/2 hr). Neither the serum FSH levels nor the BSR of FSH differed between age groups on proestrus or diestrus-1. These results show that age-associated increases in circulating FSH levels on estrus may be attributed to an enhanced basal secretion of FSH from the pituitary gland.

Different neuroendocrine mechanisms regulate the acute pituitary follicle-stimulating hormone response to orchidectomy and ovariectomy

The following experiments were conducted to determine whether a sex difference exists in neuroendocrine mechanisms controlling acute pituitary follicle-stimulating hormone (FSH) responses to castration. Adult male rats and 4-day cycling female rats on diestrus 1 were injected intraperitoneally with either phenobarbital sodium (PhB, 80 mg/kg b.w.) or vehicle at 08:00 h. Following a blood collection at 10:00 h, rats given PhB or vehicle were either sham castrated or castrated under ether. Additional blood samples were obtained, and supplemental PhB or vehicle injections were given at 3, 8, 13, 18, and 24 h after castration. Administration of PhB to male rats completely prevented acute increases in plasma luteinizing hormone (LH) and FSH levels after orchidectomy (ORDX). In contrast, PhB treatment did not prevent initial rises in plasma FSH levels at 8 h after ovariectomy (OVX) and only partially suppressed OVX-induced increases in plasma FSH levels between 13 and 24 h. Plasma LH levels were not elevated by 24 h after OVX. In order to specifically evaluate the role of LH-releasing hormone (LHRH) in mediating the PhB-sensitive rises in gonadotropins after castration, groups of male rats and female rats on estrus were injected subcutaneously with 400 micrograms of a potent LH-RH antagonist (ALHRH) or oil at 12:00 h. At 10:00 h on the next morning, an initial blood sample was taken, and all rats were castrated under ether. Additional blood samples were taken at times indicated in the previous experiment.(ABSTRACT TRUNCATED AT 250 WORDS)