Conservation of functionally important epitopes on myelin associated glycoprotein (MAG).

Phylogenetic conservation of protein domains often points to functionally important regions. As a step toward mapping these sites on myelin associated glycoprotein (MAG) we have determined the species distribution of epitopes recognized by a panel of anti-MAG antibodies (Ab). Monoclonal antibodies (mAb) B11F7, GenS3 and 28 recognized MAG only in mammalian species. However, the mAb 513 which inhibits MAG binding recognized a conformational epitope in a wider distribution of species including, human (Homo sapiens), bovine (Bos taurus), rat (Rattus norvegicus), chicken (Gallus gallus), quail (Coturnix coturnix japonica), lizard (Iguana iguana), snake (Thamnophis sirtalis), frog (Xenopus laevis) and turtle (all tetrapods) but not in goldfish (Crassius aurata) (a teleost). However, only MAG from mammals was shown to bind rat dorsal ganglion neurons (DRGs) suggesting that structures additional to those recognized by mAb 513 must be involved in function. Antibody 28, on the other hand, recognized only MAG species which bound to neurons, suggesting that this epitope, in comparison with mAb 513, more closely represented the functionally important region of MAG. Observed species differences in glycosylation of MAG may be functionally significant. A newly developed polyclonal Ab against MAG recognized the protein in tetrapods and teleosts, but not chondricthyes. The results show that MAG is present in a wide spectrum of species.

Initial characterization of factors from testicular fluid which alter in vitro androgen secretion by normal rat Leydig cells.

It is known that testicular interstitial fluid (TF) contains thermolabile factors that can alter in vitro production of androgens by the Leydig cells. The net stimulatory activity of this fluid increases in association with the disruption of spermatogenesis. The identity of the active agent(s) in TF is not known. Therefore, the authors used gel-liquid chromatography to initially characterize TF from control and bilaterally cryptorchid animals. The stimulatory activity of TF was retained on Concanavalin A Sepharose columns. Gel filtration on Ultrogel AcA 44 suggested a molecular size between 40 and 90 kD. The unfractionated fluid from control and bilaterally cryptorchid rats, as well as the chromatographic fractions containing stimulatory activity, were further resolved by SDS-PAG electrophoresis. At least three bands representing glycoproteins with apparent molecular size between 57 and 75 kD were seen in all samples containing stimulatory activity. No difference in the pattern of protein bands was seen between TF from control and bilaterally cryptorchid testes. However, samples reduced with beta-mercaptoethanol showed protein bands with apparent molecular size of 78 and 118 kD which were present only in unpurified control TF. These data support the possibility that the stimulatory substance in TF from control and bilaterally cryptorchid testes is a glycoprotein with a molecular size between 57 and 75 kD. Differences in the bioactivity of the unfractionated TF may be due in part to the presence of additional larger protein molecules in the control TF.

Disruption of spermatogenesis is associated with decreased concentration of immunoreactive arginine vasopressin in testicular fluid.

We have previously shown that testicular fluid contains factors that can inhibit luteinizing hormone (LH)-stimulated androgen production by Leydig cells, and others have reported the presence of immunoreactive vasopressin (iAVP) in the testes as well as in vitro inhibition by vasopressin of Leydig cell-androgen production. In the current report, we have used an established radioimmunoassay (RIA) to measure the concentration of iAVP in testicular fluid and have related changes in iAVP concentration to disruption of the seminiferous tubules. Spermatogenesis was disrupted in adult rats by surgically establishing bilateral cryptorchidism. The concentration of iAVP decreased progressively from 349 +/- 52 to 61 +/- 5 pg/ml during 4 wk. When cryptorchidism was unilaterally established, the concentration of iAVP in fluid from that testis decreased to 116 +/- 19 pg/ml while the concentration of iAVP in the contralateral scrotal testis remained unaffected. Unilateral ligation of the ductuli efferentes also caused an equivalent unilateral decrease in iAVP to 110 +/- 15 pg/ml. The osmotic pressure of the testicular fluid was not altered by disruption of gametogenesis, and the extracellular "albumin space" was not increased. Therefore, the decrease in concentration of iAVP was probably not due to dilution with increased amounts of interstitial fluid. We conclude that the disruption of spermatogenesis is associated with a decrease in the concentration of iAVP in testicular fluid and suggest that AVP or a similar peptide may be involved in the intratesticular mechanisms associated with increased production of androgen by Leydig cells after disruption of spermatogenesis.

The effect of spermatogenic disruption on the ability of testicular fluid to stimulate androgen production by normal Leydig cells.

Reports from this and other laboratories have concluded that unilateral disruption of spermatogenesis induces a predominantly ipsilateral increase in the responsiveness of Leydig cells to stimulation with luteinizing hormone (LH) and have suggested that if such effects were mediated by locally produced hormones then such "factors" should be detectable in testicular interstitial fluid. We sought to demonstrate such factors in testicular fluid from gonads subjected to a variety of treatments that disrupt gametogenesis. Fluid (TF) was drained from testes of adult rats that had been sham treated, irradiated, or treated with busulfan in utero, made unilaterally or bilaterally cryptorchid, or were unilaterally or bilaterally efferent-duct-ligated. Leydig cells obtained from normal rats basally produced 8 +/- 1 ng androgen/10(6) Leydig cells/2 h and, when maximally stimulated with LH, produced 66 +/- 3 ng. The addition of the various TFs to the incubations significantly increased both basal and LH-stimulated androgen production. TF from lesioned testes was more effective in increasing androgen production than TF from control rats. Unilateral lesions caused an increase in the ability of TF from the disrupted testes to increase the androgen production by normal Leydig cells, as compared to TF from contralateral testes. Thus, locally produced "factor(s)" do appear to modify Leydig cell function. Additional studies using TF from control and bilaterally cryptorchid animals suggest that the ""factor'' in TF is heat-labile; has a molecular size between bovine serum albumin and ovalbumin; exerts a portion of its action independently of cAMP formation; and does not appear to be LH, follicle-stimulating hormone, prolactin, or gonadotropin-releasing hormone.

A comparison of Leydig cell function after unilateral and bilateral cryptorchidism and efferent-duct-ligation.

Surgical induction of cryptorchidism or ligation of the efferent ducts disrupts spermatogenesis. The response of Leydig cells to disrupted gametogenesis was studied in vitro in tissue and collagenase dispersed Leydig cells obtained from the testes of rats that were made unilaterally or bilaterally cryptorchid or had been efferent-duct-ligated. Four wks after surgery, androgen secretion per mg of tissue or per Leydig cell in response to maximal luteinizing hormone (LH) stimulation was greater in tissue from damaged than from sham-operated testes. It was concluded that disruption of spermatogenesis resulted in Leydig cells that were hyperresponsive to LH stimulation in vitro. Unilateral lesions produced different responsiveness of Leydig cells from the testes ipsilateral and contralateral to the lesion, supporting the hypothesis that intragonadal modulation of Leydig cells function occurs when the function of seminiferous tubules is impaired. Stimulated androgen production of Leydig cells from the contralateral nonligated testis did not differ from that of the sham-operated controls. With unilateral cryptorchidism, which is accompanied by an increase in the temperature of the operated testis, Leydig cells from the scrotal testis were also hyperresponsive compared to those from sham-operated controls. This suggests a possible intergonadal influence of aspermatogenesis caused by cryptorchidism.

The effect of prenatal treatment with busulfan on in vitro androgen production by testes from rats of various ages.

Treatment of rats with busulfan in utero severely depletes the germ cell population of the seminiferous tubules. These studies have examined the in vitro capacity of testicular tissue and Leydig cells from such testes to secrete androgens. Leydig cells were identified by staining for 3 beta-hydroxy steroid dehydrogenase. Rats were studied at several ages to identify any developmental changes in the androgen-secreting capacity of control and treated gonads. At 30 days of age, no effect of treatment on serum androgen was found. At 60 and 90 days of age, treatment caused decreased androgen and increased LH content of the serum. At 12, 30, 60, and 90 days of age, the amount of androgen secreted per milligram of testicular tissue in response to LH was higher in busulfan-treated rats. Leydig cells from 60- and 90-day-old rats which had received busulfan were also hyperresponsive to LH. It was concluded that Leydig cells from testes essentially devoid of germ cells were hyperresponsive to LH. Serum androgen levels were decreased yet androgen production per Leydig cell was increased. A possible explanation of this apparent paradox is that busulfan treatment resulted in decreased numbers of Leydig cells in the gonads.

Fetal irradiation increases androgen production by dispersed leydig cells of the rat.

Damage to the seminiferous epithelium of the rat has been shown to decrease the concentration of serum testosterone. In these animals, compared to controls, the structure of the Leydig cells suggests hyperactivity. In an attempt to understand the functional changes at the Leydig cell level, we measured in vitro androgen production by whole testes, testicular fragments, and Leydig cell preparations. The androgen production of adult rats that had received 1.6 Gy of gamma radiation on day 20 of gestation was compared to that of nonirradiated controls. Leydig cells obtained from fetally-irradiated adult rats demonstrated increased basal (1.3 X) and LH-stimulated (4.4 X) testosterone production and increased hCG binding (4.8 X) per histochemically identified Leydig cell, as compared to cells obtained from nonirradiated control animals. Although the irradiated testicular tissue showed an increased responsiveness per mg/tissue compared to controls, basal and stimulated in vitro testosterone production per irradiated testis calculated from this data was diminished because of the five-fold decrease in testis size. In addition, the circulating levels of testosterone were reduced in irradiated animals. We suggest that fetal irradiation is associated with an increase of hCG binding and testosterone production per Leydig cell, and a decrease in the number of these Leydig cells per testis.