Sexual dimorphism in immunoneutralization of bioactivity of rat and ovine inhibin.

Inhibin was partially purified from bovine follicular fluid using chromatography on immobilized Procion Red 3B and anion-exchange chromatography. Ovariectomized Texel ewes were immunized against the inhibin-containing fraction from the Procion Red 3B column and the immune response was subsequently boosted with similar fractions or with the preparation obtained from the anion-exchange column. The potencies of the resulting antisera were evaluated in an in-vitro bioassay system for estimating inhibin activity, using dispersed rat pituitary cells. The antisera were found to inhibit the bioactivity of inhibin preparations from ovarian follicular fluid of bovine, porcine, ovine or human origin, as well as inhibin activity in ovine testicular lymph and rete testis fluid, in culture media from rat granulosa and rat Sertoli cells and in homogenates of rat ovaries and testes. These results indicate that the inhibin molecules from several species contain a common bioactive moiety. The results also showed that the antiserum was more effective in neutralizing inhibin activity from ovarian than from testicular sources in both sheep and rat, indicating a sex-related difference in the inhibin molecules within a species.

Improved embryo yield and condition of donor ovaries in cows after PMSG superovulation with monoclonal anti-PMSG administered shortly after the preovulatory LH peak.

Nonlactating Dutch-Friesian cows were selected from a local slaughterhouse and synchronized with Syncro-Mate B. Cows with a normal progesterone pattern were treated with PMSG (3,000 I.U. i.m.) on Day 10 followed by PG (Prosolvin 22.5 mg) 48 h later. Blood samples were collected daily and at hourly intervals from 30 h after PG. Monoclonal anti-PMSG (Neutra-PMSG) was administered i.v. at 5.8 h after the LH peak in 16 cows; controls (n = 16) did not receive Neutra-PMSG. For comparison, 16 additional cows were superovulated with FSH-P in decreasing doses, twice a day (total 32 mg), starting at Day 10. All cows were inseminated at 10 h after the LH peak. Embryos were evaluated on Days 6 and 7 after flushing upon slaughter (recovery 87%). The number of corpora lutea and follicles on the donor ovaries were counted. No significant differences in the concentrations of progesterone and LH were observed between the three superovulation groups. Upon Neutra-PMSG, PMSG in blood was completely neutralized, it was decreased to < 0.5 ug/l at AI from 7.0 ug/l at the LH peak. The number of transferable embryos was significantly higher after Neutra-PMSG (9.1 per cow) than without Neutra-PMSG (5.3). or upon FSH-superovulation (4.6). The number of cysts on the ovaries of Neutra-PMSG-treated cows was reduced similarly to that after FSH-superovulation. Treatment with Neutra-PMSG shortly after the LH peak positively affects final follicular maturation in PMSG-superovulated cows and results in a nearly two-fold increase of transferable embryos.

Yield of embryos in PMSG-superovulated cows treated with anti-PMSG six or 18 hours after the peak of luteinising hormone.

One hundred and forty-six Dutch cross Friesian cows were selected from a local slaughterhouse and synchronised with norgestomet. The 134 cows with a normal progesterone pattern after the removal of the norgestomet implant were treated intramuscularly with 3000 iu pregnant mare's serum gonadotrophin (PMSG) on day 10 followed by 22.5 mg prostaglandin 48 hours later. Blood samples were collected daily and at hourly intervals from 30 to 54 hours after the prostaglandin. The 113 cows with a pre-ovulatory peak of luteinising hormone (LH) were divided into three groups: 37 control cows (group 1) received a placebo six hours after the LH peak; 42 cows (group 2) received anti-PMSG six hours after the LH peak and 34 cows (group 3) received anti-PMSG 18 hours after the LH peak. All the cows were inseminated 10 hours after the LH peak. Six or seven days after insemination the cows were slaughtered and the embryos were evaluated after flushing the ovaries, and the numbers of corpora lutea, cysts and follicles on the donor ovaries were counted. Treatment with anti-PMSG had no significant effect on the numbers of corpora lutea or the numbers of embryos compared with the control group. The mean (+/- sem) numbers of corpora lutea were 14.7 +/- 1.4, 16.3 +/- 1.4 and 16.6 +/- 1.4 for groups 1, 2 and 3, respectively. The numbers of transferable embryos were 3.5 +/- 0.6, 4.1 +/- 0.7 and 5.0 +/- 0.7 for groups 1, 2 and 3, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

The brain-pituitary-gonadal axis in the rainbow trout, Salmo gairdneri. II. Direct effect of gonadal steroids on the gonadotropic cells.

In a cytophysiological study it was investigated whether in juvenile trout gonadal steroids stimulate the gonadotropic (GTH)-cells directly or indirectly via the brain. Pituitaries of donor animals were transplanted into the caudal musculature of testosterone-treated and non-testosterone-treated host fish. Testosterone treatment caused an increase in GTH-content in the in situ pituitaries and in the grafts. Accordingly, the gonadotrops displayed ultrastructural changes such as the appearance of well-developed Golgi systems and large globules. The stimulation of the morphological development of gonadotrops and of synthesis and storage of GTH in the allografted pituitaries indicates that testosterone affects the GTH-cells directly. In untreated juvenile trout the gonadotropin content of the pituitary and the gonadotropin concentration in the plasma vary with the time of year. This variation and the role of testosterone and gonadotropin-releasing hormone on the release of GTH are discussed.

The brain-pituitary-gonadal axis in the rainbow trout, Salmo gairdneri. III. Absence of an inhibiting action of testosterone on gonadotrophin release in juveniles.

In juvenile rainbow trout the effects of exogenous testosterone and of a synthetic gonadotrophin-releasing hormone (GnRH) on the secretion of gonadotrophin (GTH) were investigated. Treatment with implanted testosterone resulted in an accumulation of GTH in the pituitary, but did not affect the concentration of GTH in the plasma. After the testosterone implants were removed, the levels of testosterone in the circulation dropped to undetectable or control values, but the concentration of GTH in the plasma did not increase. These results indicate that testosterone stimulated the synthesis and storage of GTH, and did not prevent the release of this hormone. The synthetic GnRH, des-Gly10[D-Ala6]LH-RH ethylamide (LH-RHa) stimulated the release of GTH when injected into testosterone-pretreated fish, indicating that accumulated GTH is present in a releasable pool.

Immunocytochemical evidence for hypophysial projections of the cells of the nucleus lateralis tuberis pars lateralis in the rainbow trout (Salmo gairdneri).

In the rainbow trout the pars lateralis is the most prominent part of the nucleus lateralis tuberis (NLT). To demonstrate a morphological relationship between this lateral part of the NLT and the pituitary, immunocytochemistry was applied as a staining method. Experiments were carried out on glutaraldehyde-picric acid-acetic acid-fixed brain sections of mature male and female rainbow trout using the peroxidase-anti-peroxidase immune technique with an antiserum against 27-S-methylglucagon as the first antibody. Most of the cells in the NLT/pars lateralis reacted with the antiserum. Axons from these cells enter the pituitary, extending exclusively in the numerous neurohypophysial digitations in the pars intermedia. No immunoreactive neurohypophysial protrusions were found in those parts of the adenohypophysis where the gonadotropic cells are located, indicating that the lateral part of the NLT is not directly involved in the control of gonadotropin secretion. In addition to cells of the NLT/pars lateralis only prolactin cells in the rostral pars distalis of the adenohypophysis reacted with the antiserum used.

Accumulation of glycoprotein gonadotropin in the pituitary of juvenile rainbow trout in response to androgens and C21-steroids, including 11-steroids.

Effects of steroids on the accumulation of glycoprotein gonadotropin (GTH) in pituitaries of juvenile trout were investigated by means of scanning cytophotometry applied to immunocytochemical preparations, and with the use of a radioimmunoassay. Effects on other aspects of GTH-cell activity were analyzed by measuring the size of the gonadotrops and their nuclei. Progesterone added to aquarium water and methyltestosterone incorporated into the food showed a pronounced stimulatory effect on the accumulation of GTH. To a lesser extent, treatment with cortisol, cortisone, and desoxycorticosterone acetate administered to aquarium water, and 11 beta-hydroxy-androstenedione added to the food resulted in an increase of the hypophysial content of GTH. Steroids stimulating the accumulation of GTH in the pituitary also exhibited a positive effect on GTH-cell activity as indicated by an increase in the size of gonadotropic cells. Progesterone incorporated into the food did not influence the GTH-content and the GTH-cell activity. It is suggested that the route of administration of an exogenous steroid is essential for its effect on GTH cells in trout. Comparison of GTH values reveals an excellent correlation between the data from the radioimmunoassay and those from the corresponding densitometric measurements. No correlation was observed between values of morphometrically determined GTH-cell activity and the densitometric values reflecting hypophysial GTH content.

Quantitative estimation of pituitary gonadotropin in juvenile rainbow trout with scanning cytophotometry applied to immunocytochemical preparations.

A densitometrical method with computer-controlled scanning cytophotometry has been developed to quantify the amount of gonadotropin (GTH) in immunocytochemically treated paraffin sections of pituitaries of juvenile rainbow trout. Results obtained with this method totally correspond with those established with a radioimmunoassay of pooled pituitaries. Scanningcytophotometry offers the advantage that GTH-content of individual pituitaries of juvenile or small adult fish can be investigated.

Fine structure and function of isolated gonadotropic cells as revealed from pituitaries of immature rainbow trout, Salmo gairdneri, by means of a new enzymatic dispersion technique.

A procedure has been developed for dissociating pituitary glands of juvenile rainbow trout, Salmo gairdneri, producing a preparation of single dispersed pituitary cells in which morphological and functional integrity is preserved. The pituitaries are dispersed by sequential treatment with 0.1% collagenase, 0.04% ethylene-diamine-tetra-acetic acid (EDTA) and 0.125% dispase. The cell yield is 0.3--0.35 x 10(6) cells per pituitary with a cell viability percentage of 95 +/- 1% and single cell percentage of 87 +/- 4%. The isolated cells are kept in a suspension system and the gonadotropic cells are identified by the double antibody immuno-enzyme-cytochemical technique using anti-carp-beta-gonadotropin as first antibody. Secretory activity is estimated by measuring the gonadotropin content in cells and culture media by radioimmunoassay. Isolated cells show an autonomy of gonadotropin secretion. 17 alpha-Methyltestosterone both in vivo and in vitro stimulates the production of gonadotropin in the cells and seems to inhibit its release from the cells. It is concluded that this in vitro system can be used as a model for studying the control of gonadotropic cells in juvenile rainbow trout.

Effect of 17 alpha-methyltestosterone, estradiol-17 beta and synthetic LHRH on production of gonadotropic hormone in pituitaries of rainbow trout (organ culture).

Pituitary glands from 6-month-old sexually immature female rainbow trout, Salmo gairdneri, were kept in organ culture for 48 or 72 h. Certain groups of pituitaries were cultivated for 48 h on either control medium or medium with 17 alpha-methyltestosterone (MT), or with estradiol-17 beta (E2) in concentrations of 8.5 X 10(-7) M. Other groups of pituitaries were cultivated for 72 h on control medium, or for 48 h on either control medium or MT-medium or E2-medium, and subsequently for 24 h on medium with synthetic LHRH in concentrations of 8.5 X 10(-7) M and 8.5 X 10(-10) M. Gonadotropic (GTH) cells are identified by Alcian Blue-Periodic Acid Schiff-Orange G staining and the double-antibody immunoenzyme-cytochemical technique using anti-carp beta GTH as the first antibody. A quantitative histological procedure was used to study the nuclear size of the GTH cells in response to the different hormones. Secretory activity was estimated by measuring the gonadotropin (GTH) content in extracts of pituitaries, plasma, and the culture media every 24 h by radioimmunoassay. Cultivation on MT- or E2-enriched medium results in an increase of the total amount of GTH in the pituitary and medium, an accumulation of GTH in GTH-cells (approximately 20 percentage points) and an increase in their nuclear size, indicating a stimulation of GTH synthesis. However, autonomous GTH-release is not affected by these steroids. Subsequent cultivation of the pituitaries for 24 h with LHRH causes stimulation of GTH synthesis (approximately 20 percentage points). Preincubation with steroids increases the GTH synthesis capacity of LHRH only when used in a concentration of 8.5 X 10(-10) M. Moreover, 8.5 X 10(-7) M LHRH causes a stimulation of GTH-release. Preincubation of the pituitaries with steroids increases the responsiveness of GTH-cells to LHRH. It is concluded that GTH-production in pituitaries of immature female rainbow trout can be directly influenced by gonadal steroids and by a hypophysiotropic substance.

The brain-pituitary-gonadal axis in the rainbow trout, Salmo gairdneri: gonadal hormones and the maturation of gonadotropic cells.

Intact and castrated juvenile male rainbow trout (Salmo gairdneri) were treated with testosterone and gonadotropic hormone (GTH) to determine the maturational effects of these hormones on the GTH-cells. Electron-microscopic studies of the GTH-cells revealed that GTH and testosterone in intact animals, and testosterone in castrated fish, caused GTH-cell maturation: These cells now displayed the same appearance as GTH-cells in adult trout, including the presence of globules, a well-developed Golgi apparatus and rough endoplasmic reticulum, all of which were absent in GTH-cells of control animals. Animals with stimulated GTH-cells also had an increased GTH content of the pituitary; release of GTH could not be demonstrated. Animals treated with GTH exhibited an accelerated development of the testes, resulting in complete gametogenesis and elevated plasma testosterone levels. These results indicate that exogenous steroids as well as endogenous gonadal steroids can stimulate the full development of GTH-cells and accelerate GTH synthesis. The significance of this stimulating effect of the gonadal hormones with respect to the development of the brain-pituitary-gonadal axis and the onset of puberty is discussed.