Changes in serum immunoreactive inhibin-alpha during photoperiod-induced testicular regression and recrudescence in the golden hamster.

Serum immunoreactive inhibin-alpha (irI alpha), FSH, LH, and testosterone (T) were measured in male golden hamsters during short-day-induced testicular regression and during testicular recrudescence following the transfer from short to long days. Serum FSH levels were maximally suppressed within 2 wk of transfer to short days. In contrast to FSH, irI alpha levels were not fully reduced until after 6 wk of exposure to short days, closely paralleling the timing of testicular regression. LH and T levels were also reduced within two 2k, with maximal suppression observed between 6 and 8 wk. Conversely, when males with regressed testes were transferred to long days, serum FSH rose to peak (25 ng/ml) levels by 3 wk and then declined to usual long-day levels. In contrast, serum irI alpha levels rose gradually, reaching adult long-day levels following 10 wk of exposure. Serum LH and T levels rose to peak levels between 5 and 8 wk before declining to adult levels. FSH, LH, and irI alpha levels were also measured after castration in male hamsters maintained on long or short days. Twenty-four hours after castration, levels of irI alpha were reduced in long-day males to levels comparable to those observed in castrated short-day males. Serum irI alpha levels respond slowly to abrupt changes in FSH levels after transfer to either long or short days, suggesting that testicular irI alpha secretion may not be directly and immediately influenced by circulating FSH levels in the hamster.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental hormonal profiles accompanying the neonatal hypothyroidism-induced increase in adult testicular size and sperm production in the rat.

Neonatal treatment with the reversible goitrogen 6-N-propyl-2-thiouracil (PTU) results in a near doubling of testicular size and a 25% increase in the efficiency of spermatogenesis, without affecting circulating testosterone (T) levels in adult rats. The objectives of the present study were to examine the effects of neonatal PTU treatment on the pattern of testicular growth and circulating levels of anterior pituitary (FSH, LH, PRL, GH, and TSH), gonadal [immunoreactive inhibin-alpha (irI alpha) and T], and thyroid (T3 and T4) hormones over the first 100 days of life. Treatment of rats with PTU from birth to 24 days of age significantly reduced testicular weights between 10 and 60 days of age. However, the duration of testicular growth was extended in treated males, resulting in a 68% increase at 100 days of age. Serum gonadotropin levels in treated males were reduced throughout the experimental period, typically remaining between 50-70% of control levels. The characteristic robust prepubertal FSH peak was absent in PTU-treated males. Initially high until 20 days of age, irI alpha levels characteristically declined to adult levels (200-300 pg/ml) in control males. In treated males, irI alpha levels were reduced during the period of hypothyroidism, increased between 30 and 60 days, and then declined, but remained significantly higher (1.7- to 2-fold greater) than those observed in control males. Serum T levels were similar in treated and control males. Control males demonstrated increased T levels beginning at 45 days of age, earlier than observed in treated males; however, similar peak T levels were observed in all males. PTU treatment significantly suppressed serum GH and PRL and led to a 14-fold increase in circulating TSH during the period of treatment. However, unlike the gonadotropins, these hormones returned to control levels after PTU treatment, suggesting that the reduced levels of FSH and LH observed are not due to a generalized reduction in pituitary function. Serum T4 and T3 levels returned to control levels within 15 days after the removal of PTU. These results demonstrate that the neonatal PTU treatment-induced increases in adult testicular size and sperm production were not due to increased levels of FSH at any point in development. On the contrary, the observed increases occur in spite of chronically reduced FSH levels.

Nonsteroidal signals originating in the gonads.

The discovery of the various peptide factors in the gonads followed different paths. A number of factors were specifically searched for because of physiological experiments that predicted that an activity from the gonads was necessary to explain phenomena. Such was the case for gonadal steroids and for such peptide factors as inhibin, MIS, OMI, FRP, seminal plasma inhibin, relaxin, PA factor and other proteases, and ABP. In the process other factors such as activin and follistatin were serendipitously discovered. A second group of factors was discovered because in vitro experiments of various combinations of gonadal cell types failed to replicate in vivo findings, suggesting missing signals. Such substances are the panoply of growth factors aiding in differentiation and growth promotion and inhibition: LS and LI, P-Mod-S, clusterin, and various components of the ECM. Finally, and most recently, another set of peptides has been identified because immunological or molecular probes have been used to search gonadal tissue for factors originally discovered elsewhere; these include POMC, GnRH-like peptide, oxytocin, AVP, angiotensin, ANF, CRF, neural peptides, and c-mos. Our understanding of the relationship of most of these peptides to the local signals necessary for gonadal function is still very elementary. Clearly some like relaxin and inhibin function as important hormones, and ABP, for example, probably functions importantly in transporting testosterone down the tubule. Most local paracrine or autocrine peptide signals appear to act in relationship to gonadotropin levels probably in local differentiation in the process of gamete maturation, but this is only conjecture at this point. No experimental verification that any of these factors is involved in follicle selection for recruitment or for atresia is yet available. For many of the factors local receptors have not yet been identified. The richness of the variety of peptides in the gonads suggests that microanalysis of cell-cell signaling would be rewarding, but at the time of this writing such investigations are not yet possible.

Changes in serum immunoreactive inhibin and follicle-stimulating hormone during gonadal development in male and female rats.

We have measured serum and ovarian immunoreactive inhibin alpha (irI alpha) and serum FSH in fetal and neonatal rats from 20 days of gestation until 40 days of age. For animals aged 10 days or older, serum measurements were made on intact and gonadectomized animals. Serum irI alpha was detectable in intact male and female rats at all ages studied. In females, irI alpha levels were low until Day 5 and then increased steadily to peak at Day 25. Thereafter they declined until Day 35 to reach levels typical of adult females. There was a significant decrease in irI alpha levels 24 h after ovariectomy at all ages. Serum FSH levels in females were low until Day 7, then increased rapidly to plateau from Days 10-15. The levels then declined until Day 25 and were generally unchanged after that time. There was a significant increase in FSH 24 h after ovariectomy in rats aged 20 days and older, and in younger rats by 48 h after ovariectomy. In male rats, serum irI alpha levels were significantly higher than females until Day 7. The levels increased at Day 7 and then remained relatively constant until Day 20, after which they declined to reach typical adult male levels. Serum irI alpha levels were significantly lower in males than females from Days 25-40. There was a significant decrease in serum irI alpha 24 h after castration at all ages studied. Serum FSH levels in males were low until Day 20, increased at Day 25, and thereafter remained relatively unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Circulating radioimmunoassayable inhibin during periods of transient follicle-stimulating hormone rise: secondary surge and unilateral ovariectomy.

We have measured changes in circulating immunoreactive (ir-) inhibin in male and female rats using an RIA with an antiserum raised against porcine inhibin alpha (1-26)-Gly-Tyr. The same synthetic peptide was used for standards and for the preparation of tracer. Serum ir-inhibin levels were significantly higher in intact female than in intact male rats (p less than 0.001). Immunoreactive inhibin was significantly reduced in both sexes 24 h after bilateral gonadectomy (p less than 0.0001). Unilateral ovariectomy (ULO) of female rats on metestrus caused a transient decrease in serum inhibin 8 h after surgery, but levels were not significantly different from those of sham-operated controls at later times after surgery. Increases in serum FSH and LH were observed for 8-18 h after ULO. Serum ir-inhibin levels were also measured on the early morning of estrus during the secondary FSH surge. At this time, ir-inhibin levels were low, while FSH levels were high and LH levels were low. These results show that serum ir-inhibin levels in rats are decreased at times when serum FSH levels are high.

Partial purification and characterization of a novel growth hormone-releasing factor (GRF) from teleost brain related to the rat hypothalamic peptide.

A growth hormone-releasing factor (GRF)-like molecule has been partially purified and characterized from acid extracts of codfish (Gadhus morhua) brain using immunoaffinity and gel chromatography, followed by HPLC. This material has a mol.wt. which is similar to known mammalian forms of GRF but is immunologically and/or chromatographically distinct from previously described GRF peptides. However, it is related to rat(r) GRF(1-43) since it causes marked displacement in the rGRF RIA. Codfish GRF is a highly specific and potent hypophysiotropic factor as shown by its ability to stimulate the release of GH, but no other hormone, from rat anterior pituitary cells in vitro. These findings suggest that, phylogenetically, GRF is an ancient molecule with its biologic activity and certain immunoreactive domain(s) conserved, at least, from teleost to mammal.

Molecular forms of gonadotropin-releasing hormone associated peptide (GAP): changes within the rat hypothalamus and release from hypothalamic cells in vitro.

We have developed RIAs using antisera directed against the cryptic peptide of the GnRH precursor (termed GnRH-associated peptide, GAP) and have used these together with a GnRH assay to characterize proGnRH-derived peptides in rat hypothalamic extracts. On Sephadex chromatography we have identified three molecular forms of GAP-like immunoreactivity (GAP-LI), in addition to the GnRH decapeptide. The largest of these forms is an 8.0-kilodalton (kD) GAP-LI which appears to be the complete proGnRH peptide. The second is a 6.5-kD GAP-LI, and is similar to the complete cryptic peptide (i.e. proGnRH14-69 or GAP1.56). The third peptide is a 2.5 kD C-terminal fragment of the cryptic peptide, representing a processed form of GAP. In whole hypothalamic extracts from normal rats the 8.0-kD form was the major form, comprising 60-70% of the total GAP-LI. All three forms were present in three distinct areas of the rat hypothalamus, namely median eminence (ME), anterior and mid-hypothalamus. However in the ME the proportion of 8.0-kD GAP-LI was significantly reduced and the proportion of 6.5-kD GAP-LI significantly increased compared to anterior and mid-hypothalamic samples (p less than 0.05). In whole hypothalamic extracts from pregnant and lactating rats the total content of proGnRH-derived peptides was reduced but the relative proportions of these peptides were not significantly changed from normal female rats. However, in postlactating rats, 2 weeks after removal of pups, the total levels of GAP-LI were unchanged compared to normals, but the percentage of 8.0-kD GAP-LI was significantly decreased and the percentage of 2.5-kD GAP-LI significantly increased compared to normals (p less than 0.05), suggesting that proGnRH may undergo additional processing dependent on physiological condition. In fetal and neonatal rats the proportion of the 6.5-kD peptide was increased and that of the 8.0-kD peptide decreased compared to adults, and this change became less significant with increasing age. In ovariectomized rats the proportion of 6.5-kD GAP-LI was increased and that of 8.0-kD GAP-LI decreased; this change was partially reversed with steroid treatment. Both the 6.5 and 2.5-kD forms were released by high K+ stimulation of neonatal hypothalamic cells in culture. These results indicate that there is differential processing of the proGnRH precursor within the hypothalamus and in altered physiological states.

Purification and characterization of luteinizing hormone-releasing hormone from codfish brain.

We have purified luteinizing hormone-releasing hormone (LH-RH) from codfish brain and have demonstrated its identity with salmon LH-RH (sLH-RH). An antiserum raised against sLH-RH was used in a specific radioimmunoassay (RIA) to monitor purification and to manufacture an immunoaffinity chromatography column for the initial purification step. The cross-reactivity of the sLH-RH RIA with mammalian LH-RH was 0.1%. Acid extracts of codfish brains were sequentially purified by immunoaffinity chromatography, gel-filtration chromatography, and three steps of reverse-phase HPLC. The purified material and synthetic sLH-RH coeluted on reverse-phase HPLC and exhibited similar biological activity in a dispersed pituitary cell bioassay. Furthermore, the amino acid composition of the purified material was identical to salmon LH-RH. These results suggest that there is structural conservation of LH-RH between these species of teleost fish.

Corticotrophin-releasing factor-like immunoreactivity and bioactivity of human fetal and adult hypothalami.

Corticotrophin releasing factor-like immunoreactivity (CRF-LI) and bioactivity, and arginine vasopressin-like immunoreactivity (AVP-LI) have been measured in extracts of human fetal and adult hypothalamic tissue and their development with the gestational age of the fetuses (12-27 weeks) studied. CRF-LI was measured by a radioimmunoassay developed for ovine corticotrophin-releasing factor (oCRF-41). Corticotrophin-releasing factor bioactivity was measured in a rat isolated anterior pituitary cell perfusion system. CRF-LI and bioactivity and AVP-LI were all detectable in fetal hypothalamic extracts from 12 to 13 weeks of gestational age. CRF-LI was also present in human fetal pituitary glands from 12 weeks of gestational age. The concentration of CRF-LI in the fetal hypothalamic extracts (9.2 +/- 11.4 ng/g, mean +/- S.E.M., n = 33) showed no significant correlation with the gestational age of the fetuses. However the concentration of AVP-LI (25.0-36.8 ng/g, n = 17) did show a positive correlation (r = 0.508, P less than 0.05) with gestational age, as did the concentration of CRF bioactivity (471.3-556.3 ng ACTH released/g tissue, n = 13, r = 0.725, P less than 0.01). The CRF bioactivity of all fetal hypothalamic extracts was potentiated by the addition of synthetic human (h)AVP, but the bioactivity of the adult hypothalamic extracts was not, presumably because of the higher levels of AVP-LI already present in the adult extracts. Pretreatment of tissue extracts with antisera to oCRF-41 and/or hAVP reduced the CRF bioactivity of all hypothalamic extracts. Sephadex chromatography of fractions which co-eluted with synthetic oCRF-41 or hAVP contained CRF bioactivity and this bioactivity was potentiated when synthetic hAVP or oCRF-41, respectively, were added to the fractions. However, a larger molecular weight form of CRF-LI (8000-10 000 daltons), which was observed only in fetuses of 20 weeks of gestational age or less, did not contain any significant CRF bioactivity.

Corticotrophin-releasing factor--mediated adrenocorticotrophin release from rat anterior pituitary cells is potentiated by C-terminal gastrin-releasing peptide.

The remission of Cushing's syndrome following surgical removal of a tumour containing bombesin-like immunoreactivity (BLI), but insignificant levels of ACTH, is described. However, an acid extract of the tumour tissue caused the release of ACTH from isolated rat anterior pituitary cells in vitro. These observations led to an investigation of the effects of synthetic C-terminal gastrin-releasing peptide (GRP(14-27] on ACTH release from isolated rat anterior pituitary cells. GRP(14-27) (10-1000 ng/ml) directly stimulated the release of ACTH in vitro, whereas lower doses (10-1000 pg/ml), ineffective themselves in eliciting ACTH release, potentiated the CRF-mediated in-vitro release of ACTH.