Endocrinologic and clinical evaluation following a single administration of a gonadotrophin-releasing hormone agonist (Zoladex), in a depot formulation, to premenopausal women.

A single 3.6-mg Zoladex subcutaneous depot injection was effective in suppressing the pituitary-ovarian function for about 5 weeks in nine regularly menstruating, premenopausal volunteers. Menses returned approximately 9 weeks after the injection. Zoladex depot is a novel approach in the administration of GnRH agonists and offers great therapeutic potential.

Separate ontogeny of two macrophage-like accessory cell populations in the human fetus.

Human macrophage-like accessory cells were analyzed as they emerge in the absence of extrinsic antigens during fetal development. Monoclonal antibodies to monocytes/macrophages were used in combination with antibodies to HLA class II molecules. In the yolk sac and mesenchyme sampled at wk 4 to 6 of fertilization age, cells with dendritic morphology formed two populations distinguishable by phenotypic criteria: type i (majority) carried both macrophage-associated (RFD7+) and monocyte-associated markers (UCHM1+) but no detectable HLA-DR antigen, and type ii (minority) constitutively expressed class II (HLA-DR and -DP) but no RFD7 and UCHM1. The emergence of this heterogeneity preceded the formation of both thymus and bone marrow. During additional development, type i and type ii cells seeded to different microenvironments and underwent some additional phenotypic changes. Cells of type i, the RFD7+ population with high lysosomal (acid phosphatase) activity, were seen in the thymic cortex, marginal zone of lymph nodes, splenic red pulp, and in the midst of erythropoietic activity within the bone marrow. These cells were UCHM1- and class II-. Cells of type ii formed the population of HLA-DR+, RFD7- interdigitating cells, early inhabitants of T cell areas in the developing thymic medulla, lymph nodes, spleen, and tonsil. The Type ii cells that had already settled in their nichès expressed not only HLA-DR and -DP but also HLA-DQ, and another class II antigen identified by the antibody RFD1, which shows the restricted tissue distribution of HLA-DQ, but is governed by genes that are outside of and telomeric to the HLA-DQ region (or HLA-DR). Finally, subpopulations of macrophages (RFD7+, acid phosphatase-positive) in the fetal gastrointestinal and hepatic systems were HLA-DR+; the latter appear to include precursors of Kupffer cells in the developing liver.

Human B cell development. II. Subpopulations in the human fetus.

In man, during fetal development the B cell populations show distinct phenotypes at different tissue sites. The pre-B and B lymphocytes of the fetal liver and bone marrow express IgM and B cell markers, B1 (CD20) and BA-1 (CD24). These "early" cells are negative with a number of other reagents, anti-IgD, RFB4 (CD22), RFB6 (CD21), and RFA-2, which on the other hand recognize peripheral B cells. These peripheral B lymphocytes in the developing fetus are heterogeneous. The diffusely distributed B cells in the earliest lymph node samples, 16 to 17 wk of gestational age, and from 16 to 21 wk in the spleen, are strongly IgM+ (IgD+,RFB4+,RFB6+, and RFA-2+) but lack T cell-associated markers such as T1 (CD5, p 67,000 dalton equivalent of murine Ly-1) and Tü-33. In fetal lymph nodes, primary nodules develop around the follicular dendritic (FD) cells from 17 wk onward, and contain a virtually pure population of B cells; B1+,BA1+,RFB4+,RFB6+,RFA-2+, which simultaneously express IgM,IgD together with T1 (CD5), a T cell-associated antigen. A sizeable subpopulation of these IgM+,T1+ cells are also positive for Tü-33, another T cell-associated marker. In the spleen, the B cells of the IgM+,IgD+,T1+ type appear in smaller numbers and only relatively late around wk 22. These cells are diffusely distributed at first, and start accumulating around the small FD cell clusters as soon as these emerge about the 23rd gestational wk. At that time, the IgM+,T1+B cells can also be washed out from the peritoneal and pleural cavities. The T1+,IgM+B cells may represent the normal equivalent cells of B chronic lymphoid leukemia and centrocytic lymphoma, and appear to be the counterpart of Ly-1+,IgM+B cells in the mouse.

Identification of oxytocin and vasopressin in the testis and in adrenal tissue.

Oxytocin, vasopressin and neurophysin-like immunoreactivity have been identified and measured by radioimmunoassay in extracts of human and rat testis and human fetal adrenal tissue. The authenticity of these polypeptides has been confirmed by their behaviour on high performance liquid chromatography. The concentrations of the hormone were too great to be explained by known circulating levels of the polypeptides, and their presence in steroid secreting organs suggests a possible role for them in steroidogenesis. The peptides may be taken up and concentrated by the tissues but the co-localisation of neurophysins with the hormones points towards local synthesis.

The ontogeny of terminal deoxynucleotidyl transferase positive cells in the human fetus.

The ontogeny of cells containing the enzyme terminal deoxynucleotidyl transferase (TdT) in human fetal liver, bone marrow, and thymus has been studied using a highly specific antiserum to TdT together with monoclonal antiprecursor cell antibodies in double and triple marker immunofluorescence. TdT+ cells were first observed in fetal liver at 12 wk of gestation and accounted for 55% of the lymphoid-like cells isolated after Ficoll-Hypaque separation. TdT+ cells were first observed in the bone marrow 16 wk after gestation. Like TdT+ cells in normal infant bone marrow, the majority of TdT+ cells in fetal liver and bone marrow expressed both BA-1 and RFB-1 antigens. This suggests that fetal TdT+ cells include progenitors of the B lineage (BA-1+) and perhaps of thymocytes (RFB-1+). Nevertheless, TdT was not observed in fetal thymocytes until after 20 wk of gestation, although thymic blasts and the majority of thymocytes were strongly RFB-1+ from 12 wk of gestation. These results clearly show that fetal thymus is first populated by TdT, RFB-1+, BA-1 cells, but does not exclude the fact that a second "wave" of TdT+ prothymocytes, possibly bone marrow derived, also exists.

Oxytocin, vasopressin and neurophysins in the hypothalamo-neurohypophysial system of the human fetus.

The content of vasopressin, oxytocin and their related neurophysins was measured in the hypothalamus and pituitary gland of mid-trimester human fetuses. Vasopressin was present in both tissues approximately 3-4 weeks before oxytocin. The levels of the hormones in the pituitary gland increased 1000-fold over the next 3-4 months. During this time, the very high vasopressin/oxytocin ratio gradually deceased but did not reach unity in the period studied. In contrast, both the vasopressin-associated neurophysin and the oxytocin-associated neurophysin appeared in the pituitary gland at the same gestational age and showed the same exponential increase with fetal age. Lower levels of the neurophysins and the nonapeptides were found in the hypothalamus and the levels increased more slowly with fetal age. Our results suggest that the high vasopressin/oxytocin ratios observed in fetal life are due to differences in the rate of maturation of the hormone precursor, rather than to differences in the rate of de-novo synthesis.

A 3-dimensional reconstruction of the hypothalamo-neurohypophysial system of the rat. The neurons projecting to the neuro/intermediate lobe and those containing vasopressin and somatostatin.

A three-dimensional reconstruction of the rat's hypothalamo-neurohypophysial system, depicting the neurons that project to the neuro-intermediate lobe of the pituitary, has been made by using retrograde transport of horseradish peroxidase injected into the neuro-intermediate lobe and by using immunocytochemistry with antisera to vasopressin and somatostatin. The overall picture illustrates the neurons situated in the walls of a pair of ill-defined cone-shaped tunnels, the apices pointing anteriorly. Among the neuronal aggregates in the tunnel wall two, the paraventricular and forniceal nuclei, appear similar in shape but clearly separated by a gap of at least 150 micrometers. Many of the vasopressin-positive neurons lie in the same nuclear aggregates with two notable exceptions: the suprachiasmatic nucleus contains many vasopressin-positive cells but does not project to the pituitary, and the forniceal aggregate, which does project to the pituitary, contains no vasopressin-positive cells. Somatostatin-positive cells are situated close to the third ventricle and their size is intermediate between parvocellular and magnocellular. Cell counts show only half the cells in the system lying in the supraoptic and paraventricular nuclei, the rest being in "accessory nuclei".

Intra-axonal transport and turnover of neurophysins in the rat. A proposal for a possible origin of the minor neurophysin component.

1. Radioactivity associated with the three neurophysins in the neural lobe of the rat was determined at intervals up to 5 weeks after an intracisternal injection of [(35)S]cysteine. 2. The radioactivity associated with the two major neurophysins (one supposedly associated with vasopressin and the other with oxytocin) increased linearly for 12h after the injection and the ratio of the rates of increase in the two proteins was very similar to the ratio of vasopressin to oxytocin in the gland. 3. From 12h onwards the radioactivity associated with each major neurophysin declined exponentially but the half-life of the supposed oxytocin-neurophysin (13.3 days) was shorter than that for the supposed vasopressin-neurophysin (19.8 days). 4. The kinetics of labelling of the minor neurophysin was quite different from that of the two major ones. It became slowly labelled during 3-5 days after injection and the radioactivity hardly decreased during the following 4 weeks. 5. The data could support the hypothesis that the minor neurophysin is a metabolic product of oxytocin-neurophysin. The exponential rate of disappearance of radioactivity from oxytocin-neurophysin and the minor component taken together has a rate constant similar to that for vasopressin-neurophysin (e.g. half-life=18.9 days).

A model for the passage of the nurohypophysial hormones and their related proteins through the rat neurohypophysis.

The change in the radioactivity of vasopressin-neurophysin in the rat neurohypophysis after an intracisternal injection of [(35)S]cysteine was fitted to several mathematical models. The data fitted best a model in which there is a linear input of radioactive protein into one pool of the neurohypophysis, from which it is either released by an exponential process or transferred to a second pool from which it is released by a second exponential process with a rate constant much lower than the first. This model is compatible with the existence of a ;readily releasable' pool first postulated by Sachs et al. (1967). Data for the change in radioactivity of vasopressin also gave a good fit in this model. Calculation of the rate constants suggested that the first pool represented about 2% of the total hormone.

The number of neurophysins in the rat. Influence of the concentration of Bromophenol Blue, used as a tracking dye, on the resolution of proteins by polyacrylamide-gel electrophoresis.

1. The concentration of Bromophenol Blue used as tracking dye in polyacrylamide-gel electrophoresis affected the resolution of rat neurophysins. 2. A final dye concentration of 1mug/ml in the tris-glycine running buffer was found to give the best results. 3. The presence of two major and one minor neurophysin(s) in the rat was confirmed. 4. The two major proteins were found to re-run as single discrete bands, which had the same mobilities in the absence of dye and different mobilities in its presence.

Tentative identification of a vasopressin-neurophysin and an oxytocin-neurophysin in the rat.

1. Rat neurohypophysial extracts have been examined by polyacrylamide-gel electrophoresis. 2. Three of the proteins were tentatively identified as neurophysins by their acidic nature and their disappearance after dehydration of the animals. 3. These proteins were radioactive 24h after intracisternal injection of [(35)S]cysteine. 4. Two of the proteins were present in much greater quantities than the third, and these two were present in the gland in the same ratio as the hormones vasopressin and oxytocin. 5. One of these proteins was absent from glands of rats homozygous for diabetes insipidus but present in heterozygous animals. 6. It is suggested that these two proteins are the vasopressin-neurophysin and oxytocin-neurophysin of the rat.