Non-gonadal Tissues Research Articles

Novel expression of human chorionic gonadotropin/luteinizing hormone receptor gene in brain

LH from anterior pituitary and hCG from placenta bind to a common receptor in gonadal and nongonadal reproductive tissues. There have been numerous examples suggesting that the brain may also contain hCG/LH receptors, yet there has been no evidence for their existence so far. We now demonstrate by reverse transcription-nested polymerase chain reaction and northern blotting that the rat brain contains hCG/LH receptor mRNA. A major receptor transcript of 2.6 kilobases and minor transcripts of 1.8 and 4.4 kilobases were found. Western immunoblotting, ligand blotting, and covalent receptor cross-linking studies have shown that rat brain also contains an 80-kilodalton receptor protein that can bind hCG and hLH, but not hFSH. Rat testis used as a positive control showed a higher abundance of multiple transcripts and an 80-kilodalton receptor protein that can bind [125I]hCG. Rat liver used as a negative control did not contain any receptor transcripts or protein. In situ hybridization, dot blotting, immunocytochemistry, and topical autoradiography have revealed that hCG/LH receptors are present in rat hippocampus; dentate gyrus; hypothalamus; cerebellum; choroid plexus; ependymal cells of third, fourth, and lateral ventricles; cortex; brainstem; bovine hypothalamus; and human area postrema. These novel findings could potentially explain numerous previous observations and suggest new possibilities concerning the regulation of brain functions by hCG and LH.

A comprehensive study of genic variation in natural populations of Drosophila melanogaster. VII. Varying rates of genic divergence as revealed by two-dimensional electrophoresis.

Four sibling species from the melanogaster subgroup (Drosophila melanogaster, D. simulans, D. sechellia, and D. mauritiana) were studied for genetic divergence, by high-resolution two-dimensional protein electrophoresis (2DE) coupled with ultrasensitive silver staining. A total of eight tissues from larval and adult developmental stages representing both gonadal (germ-line) and nongonadal (somatic) tissues were analyzed for protein divergence between species. Close to 400 polypeptides (protein spots) were scored from each tissue and species, and protein divergence was measured on the basis of qualitative differences (presence/absence) of protein spots in pairwise species comparisons. The observed levels of genic divergence varied among tissues and among species. When larval hemolymph proteins (which are known to be highly polymorphic) were excluded, there was no evidence to suggest that either the larval or adult-stage proteins, as a whole, are more diverged than the other; variation between different tissues rather than between developmental stages appears to be the most significant factor affecting genetic divergence between species. The reproductive tissue (testis and accessory gland) showed more divergence than did the nonreproductive tissue; D. melanogaster testis (from both larvae and adult males) showed the highest level of divergence. In view of the previous observation that D. simulans, D. mauritiana, and D. sechellia show similar but significantly less reproductive isolation from each other than from D. melanogaster, the present results suggest a correlation between the levels of reproductive-tract-protein divergence and the degree of reproductive isolation in these species.

Localization of Inhibin Alpha-Subunit Immunoreactivity in the Rat Adrenal Cortex

Abstract Inhibin, a water soluble, non-steroidal glycoprotein hormone that inhibits follicle-stimulating hormone secretion, is produced by the gonads and several non-gonadal tissues (placenta, pituitary and brain). This study describes the presence of inhibin alpha-chain-immunoreactive cells in the adrenal cortex of intact adult male rats and the effects of hypophysectomy and adrenocorticotropin treatment on the expression of inhibin in that gland. In intact rats, the majority of the immunoreactive cells were found scattered in the zona glomerulosa and reticularis. The zona fasciculata contained only a few cells immunopositive for the inhibin alpha-chain. No immunoreactive cells were found in the medulla. Hypophysectomy resulted in a dramatic reduction in alpha-subunit immunoreactivity with only scattered cells observed in the zona glomerulosa and reticularis (0 to 2 cells/section). No immunoreactive cells were found in the zona fasciculata or the medulla. Following supraphysiologic adrenocorticotropin administration to hypophysectomized animals, the number of inhibin alpha-chain-immunoreactive cells significantly increased in each zone of the adrenal cortex. The most prominent changes were seen in the zona reticularis. Interestingly, in the adrenocorticotropin-treated animals scattered immunopositive cells were also present in the medulla. Our results suggest that inhibin-related peptide(s) exist within the adrenal gland and that the expression of these peptides is regulated by adrenocorticotropin. Inhibin-related peptide(s) may, therefore, play a paracrine and/or endocrine role in the adrenal function.

Identification and expression of boar sperm proteins in the reproductive tract that interact with antibodies in serum from an infertile woman

Serum designated as IS obtained from a young healthy infertile woman induced a head-to-head agglutination of ejaculated boar sperm. The immunoglobulin G (IgG) prepared from IS localized to the acrosomal region of the sperm head obtained from the corpus and cauda epididymis as determined by an indirect immunofluorescent method. The IgG interacted with a boar sperm protein with an estimated molecular weight of 45-kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoretic (SDS-PAGE) immunoblotting technique. However, the IgG did not interact with proteins extracted from sperm obtained from the testis and caput epididymis or from non-gonadal tissues including liver, kidney, spleen, muscle and serum. The IgG interacted with additional proteins of about 75- and 38-kDa present in the corpus and cauda epididymal fluids but not those in the caput epididymal fluid. The staining intensity of the 75-kDa band was reduced and that of the 38-kDa was nullified with ejaculated seminal plasma proteins. The interacting proteins were adsorbed when chromatographed on Concanavalin A Sepharose column, suggesting that they are glycoproteins.

GONADOTROPIN-INDUCED UP- AND DOWN-REGULATION OF RAT OVARIAN LH RECEPTOR MESSAGE LEVELS DURING FOLLICULAR GROWTH, OVULATION AND LUTEINIZATION

Induction of follicular growth by PMSG is associated with increased ovarian LH receptor content, whereas the preovulatory surge of LH decreases LH binding sites, followed by a secondary increase in receptor numbers coincident with corpora lutea formation. Based on the recently reported LH receptor cDNA sequence, we have performed a reverse transcription-polymerase chain reaction to obtain LH receptor cDNA clones and generated a 32P-labeled cRNA probe to examine the dynamic changes in ovarian LH receptor mRNA levels during gonadotropin induction of follicular growth, ovulation and luteinization. Northern blot analysis of ovary RNA revealed hybridization signals of about 7.0, 4.2, 2.5 and 1.8 kb, with no hybridization to nongonadal tissues. PMSG increased the intensity of all four LH receptor messages at 24 h, preceding an increase in LH receptor number, with peak LH receptor mRNA and receptor content observed at 52 h. Treatment with hCG resulted in decreased LH receptor binding and mRNA levels by 6 h after injection, with maximal inhibition (greater than 85%) of message at 12 to 24 h after hCG treatment. Subsequently, LH receptor message levels increased again at 3 days after hCG, concomitant with increased [125I]hCG binding. A further increase in LH receptor content, but not message levels, was observed 5 days after hCG. These results demonstrate that the induction of LH receptors by PMSG is preceded by increased LH receptor mRNA levels. Furthermore, ligand-induced down-regulation of the LH receptor following an ovulatory dose of hCG is associated with decreased LH receptor message content, followed by increases in LH receptor message levels and binding sites during subsequent luteinization. Thus, the up- and down-regulation of ovarian LH receptors during follicle growth, ovulation and luteinization is probably due, at least in part, to changes in receptor message modulation.

Gonadotropic hormone (GtH) receptors in the testis of the troutSalmo gairdneri: in vitro studies

A particulate fraction obtained from trout testis at the time of spermiation shows saturable binding sites for(125)I-labeled salmon gonadotropin ((125)I-GtH). Non-gonadal tissues (liver, muscle and spleen) did not demonstrate specific(125)I-GtH binding. The tracer's specific activity was determined by the self-displacement method (18 to 30 μCi/μg). Maximal specific binding ability of(125)I-GtH varied from 20 to 30% of the labelled ligand added, depending on the hormone preparation. Specific binding of(125)I-GtH to 20 mg of the testis membrane varied from 40 to 85% of the total binding depending on the method of membrane prepratation, and was competitively inhibited by concentrations of unlabelled GtH ranging from ca 1 to 1000 ng/ml of incubate. Gonadotropin of mammalian origin, ovine TSH or salmon prolactin competed only weakly, or not at all, for testicular gonadotropin binding sites (relative potencies s-GtH>>FSH=hCG>s-PRL>bTSH). Scatchard analysis of equilibrium binding studies shows that saturable gonadotropin binding was due to a class of high affinity binding sites (sites I Ka≊3×10(10) M(-1)) and possibly to a second class of lower affinity binding sites (sites II Ka=5 to 14×10(8) M(-1)). The binding capacity of sites I, as measured in enriched membrane preparations, was 45±18 fmoles/g of testis during the period of spermiation. The concentration of GtH required to obtain half maximal displacement of(125)I-GtH in the binding studies was of the same order of magnitude as the apparent ED50 for GtH stimulation of 11-Cetotestosterone (11KT) secretion by trout testesin vitro. Mammalian LH and FSH were 100 to 1000 folds less potent than salmor GtH to increase 11 KT secretion.

Alpha-inhibin gene expression occurs in the ovine adrenal cortex, and is regulated by adrenocorticotropin.

Inhibin is a glycoprotein hormone composed of two nonidentical subunits. It is produced by the ovary and testis and plays a vital role in gonadal function by inhibiting the secretion of FSH. More recently, additional activities associated with inhibin peptides have been identified. Inhibin heterodimers (alpha-beta) are reported to act directly on ovarian granulosa cells and inhibit estrogen production induced by FSH. Furthermore, homodimers of beta-inhibin subunits stimulate the secretion of FSH, an activity that is directly opposite to that of inhibin. Each of these inhibin-related activities are concerned with the hypothalamic-pituitary-gonadal axis. We have investigated further the complexity of inhibin activity by determining whether inhibin genes are expressed in nongonadal tissue. RNA hybridization experiments demonstrate that the alpha-inhibin gene is expressed in the sheep adrenal cortex and hybridization histochemistry shows that this gene is expressed in each of the functional zones within the cortex. Dot blot analysis showed that the level of alpha mRNA within the adrenal is influenced by ACTH, one of the major regulators of adrenal cortex function. These observations imply that there are inhibin-related peptides not directly associated with the gonads. beta-inhibin gene expression was not clearly detected in the adrenal and we conclude that if expression occurs then it does so at extremely low levels.

A genetic analysis of the sex-determining gene, tra-1, in the nematode Caenorhabditis elegans.

The normal sexes of Caenorhabditis elegans are the self-fertilizing hermaphrodite (XX) and the male (XO). The autosomal gene tra-1 is a major switch gene controlling sexual phenotype. Mutant phenotypes of 43 loss-of-function (lf) tra-1 alleles and 22 gain-of-function (gf) tra-1 alleles are described and discussed. The tra-1(lf) alleles are recessive and, in general, masculinizing. The most severe mutations (such as seven out of eight identified amber alleles) can transform XX animals into fertile males. These mutations have little effect on XO animals (which are male already) but lead to some abnormalities in XO gonadal development, indicating that tra-1 has functions in normal development of both sexes, although its major function is confined to the XX hermaphrodite. Weaker tra-1(lf) alleles lead to incomplete masculinization of XX animals, resulting in a variety of intersexual phenotypes. the tra-1(gf) alleles are dominant and have an opposite, feminizing effect. Six out of 22 can transform XO animals into fertile females or hermaphrodites, whereas the remainder cause partial feminization. All 22 transform XX animals into fertile females. Limited intragenic mapping indicates that the gene is large and that gf alleles map to a location different from lf alleles. The results suggest that the tra-1 gene has several roles in wild-type sexual development. First, tra-1 activity dictates female, as opposed to male, development in all nongonadal tissues of XX animals. Second, tra-1 activity dictates female development in the somatic gonad of XX animals. Third, a high level of tra-1 activity may act to inhibit spermatogenesis in the XX germ line, thereby assisting the switch from spermatogenesis to oogenesis in the hermaphrodite. These three functions are all feminizing and specific to the XX animal; the gene also has minor functions in the XO animal, which are to assist normal male somatic gonad development and to promote abundant spermatogenesis in males. A low level of both spermatogenesis and oogenesis can occur in the absence of tra-1 activity. Both the function and the regulation of this gene must be complex.

The occurrence of serological H-Y antigen (Sxs antigen) in the diandric protogynous wrasse, Coris julis (L.) (Labridae, Teleostei)

The serological sex-specific (Sxs) antigen (previously called ‘H-Y antigen’) has been shown, in various vertebrate species ranging from fish to mammals, to be characteristic of the heterogametic sex. We studied a protogynous hermaphrodite, Coris julis, in order to examine whether the change of a female to a secondary male also involves a change in the Sxs-antigen phenotype. The (homogametic) females of this species were found to be Sxs negative, while both primary and secondary males were Sxs positive. This was true not only for gonads but also for nongo-nadal tissues. The administration of androgen to females is known to cause sex inversion in this species; we were able to demonstrate this again at the histological level, and found that androgen results in a Sxs positive phenotype in all tissues studied (gonads, spleen, muscle). We propose that androgen is responsible, directly or indirectly, for the occurrence of the Sxs antigen.

Hormonal regulation of the peripubertal surge of insulin-like growth factor-I in the rat.

The marked increase in circulating insulin-like growth factor-I (IGF-I) levels during puberty observed in primates indicates an important functional relationship between hypothalamic-pituitary gonadal function and hormonal regulation of peripubertal circulating IGF-I levels. Recent studies demonstrating local production and secretion of gonadal peptides including IGF-I suggest that increased circulating IGF-I levels during puberty might be due to direct gonadal secretion of IGF-I or alternatively to indirect effects of increased gonadal steroid secretion on nongonadal tissues including the hypothalamus, pituitary, and liver. We therefore studied the effects of prepubertal castration on the pubertal IGF-I surge and demonstrate that castration provokes a further increase rather than ablation of the pubertal IGF-I surge in the rat. Furthermore, neonatal treatment with monosodium glutamate, a hypothalamic neurotoxin, abolishes the pubertal IGF-I surge when commenced on postnatal day 1 but not on day 5, whereas treatment with a GnRH antagonist commencing within 12 h of birth significantly reduces but does not abolish the pubertal IGF-I surge. We therefore propose that the pubertal IGF-I surge in the rat is not due to direct gonadal secretion of IGF-I or other gonadal hormones during puberty but may involve hypothalamic and/or hepatic programming by events during prenatal or very early postnatal life.

Immunohistological localization of 17 beta-estradiol and testosterone in the ovary of the rainbow trout (Salmo gairdneri Richardson) during the preovulatory period.

Antisera (AS) raised in rabbits against 17 beta-estradiol (E) and testosterone (T) were tested for their suitability to localize E and T on deparaffinized, rehydrated sections of preovulatory trout ovaries, using the unlabeled antibody technique. Conventional control experiments demonstrated the specificity of the staining reactions. Furthermore, no staining was observed after the removal of T-specific antibodies by affinity chromatography, or following gonadectomy when non-gonadal tissue sections of male trout were incubated with T-AS. Antiserum, raised against 11-oxotestosterone and devoid of antibodies cross-reacting with T, did not stain ovarian sections. The loci at which E and T are detected in the somatic compartment are consistent with the two-cell concept of estrogen synthesis, where aromatizable androgens are produced in the thecal/interstitial layer and serve as substrates for estrogen synthesis in granulosa cells. Both steroids were detected in yolk vesicles from the stage of endogenous vitellogenesis. T-AS showed affinity for nuclei of vitellogenic oocytes. Nucleoli were not stained.

A monoclonal antibody, EC-1, derived from a syngeneically multiparous mouse alters in vitro fertilization and development

A monoclonal antibody designated ‘EC-1’ was derived from a fusion of myeloma cells with lymphoid tissue from a syngeneically multiparous, but otherwise unimmunized, mouse and was selected by screening for reactivity with teratocarcinoma cells. The IgM antibody binds to the cell surface of ova, zygotes, and 2-cell embryos. Binding is not detected on the 4- or 8-cell embryo but reappears on the morula and blastocyst. EC-1 binds to the trophoblast but not to the inner cell mass of in vitro attached blastocysts and the ectoplacental cone of the peri-implantation embryo. In adult tissues, EC-1 binds to the follicular cells of the ovary, the lining epithelium of the pregnant uterus, the interstitial region of the testes and to epidydimal but not testicular sperm. In nongonadal tissues EC-1 binds to an epitope located in some, but not all, regions of connectives tissues associated with basement membrane. The antigen detected by EC-1, as expressed on teratocarcinoma-derived cell line PYS-2, is a large glycoprotein which is sensitive to reduction. EC-1 inhibits in vitro fertilization and partially inhibits in vitro development of in vitro fertilized ova. The possible implications of EC-1 binding and activity are discussed.

Effect of tamoxifen on H-Y antigen expression and gonadal development in chicken embryos

Avian species follow the ZW/ZZ system of sex determination, which the female is heterogametic and expresses H-Y (or, more appropriately,‘H-W’) antigen. We present the results of an investigation into the effects of the antiestrogen, tamoxifen, on gonadal differentiation and H-Y antigen expression in chickens. When given at doses of 0.25–2 mg per egg immediately before incubation, tamoxifen blocked regression of the right gonad in a significant number of 14-day-old female embryos. The nonregressed right gonad had a testis-like external appearance and, in some cases, contained what appeared to be spermatogenic tubules. Tamoxifen had no histologically detectable effect on the differentiation of the left ovary or the testes. In spite of tamoxifen's histological effects on right female gonads, it did not masculinize the steroidogenic capabilities of these gonads. Whether obtained from drug- or vehicletreated embryos, the left and right female gonads always contained appreciable amounts of estrogen. In contrast, testes obtained from either drug- or vehicle-treated embryos did not contain detectable amounts of estrogen. Tamoxifen reduced the H-Y antigen levels in female liver and gonads. In both left and right female gonads, the reduction was to male levels. In female livers, tamoxifen reduced H-Y antigen to levels intermediate between those of normal males and females. Thus, the expression of H-Y antigen in both gonadal and nongonadal tissue is estrogen dependent, but the dependency appears to be more stringent for gonadal tissue. Since the morphology of left gonads obtained from tamoxifen-treated females remained ovarian even though they contained levels of H-Y antigen which were indistinguishable from those contained by testes, these data indicate that ovarian differentiation can occur in the presence of male levels of H-Y antigen.

Gonadal and nongonadal zinc concentrations in mussels

Seasonal measurement of zinc levels in mussels (Mytilus edulis) from the Tyne estuary showed a small spring peak and an annual low in September. Zinc concentration was found to be significantly lower in gonadal tissue than in nongonadal tissue. Although gonadal dry weight was strongly negatively correlated with gonadal zinc concentration there was no significant relationship between gonadal dry weight and whole body zinc levels, indicating that gonadal state has little effect on zinc variability in these animals. However, a strong negative correlation was found between log gonadal zinc concentration and log nongonadal dry weight. Construction of a gonadal index (gonadal/nongonadal dry weight) shows that the seasonal peak zinc concentration in early May occurs before major spawning has taken place. It is concluded that the seasonal dynamics of zinc in this species can be explained in terms of growth of somatic rather than germinal tissue.

Regulation and cell autonomy during postembryonic development of Caenorhabditis elegans

The role of cell-cell interaction in the postembryonic development of nongonadal tissues in the nematode Caenorhabditis elegans has been explored by selective cell ablation with a laser microbeam. Examples have been found of induction and of regulation in cell lineage and fate. Regulation in which one cell precisely or partially replaces another is seen, but only in certain groups of hypodermal cells which resemble one another closely; cells which are unique are not replaced in this way. The regulation of cell form is more widespread and less restrictive.

Induction of H-Y Antigen in the Gonads of Male Quail Embryos by Diethylstilbestrol

In quails, H-Y antigen is induced by oestrogens in the gonads of the originally H-Y negative homogametic sex but not in non-gonadal tissues. This is consistent with the view that oestrogens act via H-Y antigen in the organization of the avian gonad.

Binding studies of H-Y antigen in rat tissues

The binding capacity for H-Y antigen was studied in various rat tissues of both sexes. In nongonadal tissues (liver, kidney, brain, epidermis) binding could not be demonstrated. In contrast, the gonads are able to bind exogenously supplied H-Y antigen. In the ovary, the binding capacity remains unchanged in newborn and adult animals, while in the testis, this capacity decreases with age. A receptor like that of a proteohormone is assumed to exist in the gonads but not in other tissues. In nongonadal tissues, H-Y antigen apparently is present only if the cell itself synthesizes the antigen. The H-Y antigen receptor of the gonads is not sex-specific. Thus, the primary sex differentiation depends on whether H-Y antigen is synthesized in the organism.

The testis as a secretory organ for H-Y antigen

After cultivation of dissociated rat testicular tissues, H-Y antigen is detectable in the medium; this is not the case if nongonadal male tissues are incubated. Release of H-Y antigen by testis cells is inhibited by the addition of cycloheximide. All tissues still type H-Y positive after culture. It is assumed that the testis actively secretes H-Y antigen. This assumption is supported by the finding that the amount of H-Y antigen in the epididymal fluid increases with the age of the animals.

Classification of Different Pathologic Types

Neoplasms arising in the testis are often of complex character and differ in many respects from neoplasms of nongonadal tissues. Unfortunately, certain critically important and interrelated details are not completely understood; these concern the embryology of the testis, the histogenesis of teratomatous tumors, the histogenesis, nature, and nomenclature of those neoplasms derived from primordial germ cells, and that of those neoplasms derived from nongerminal elements. Testicular neoplasms are relatively rare, comprising somewhat less than 1% of all cancers occurring in males and having an average annual incidence of about 2.3 per 100,000 males in English speaking populations.^8,9Nevertheless, they have provoked considerable interest because more than 90% of these tumors prove to be malignant and because they occur predominantly in young men who are in the prime of life (during the third and fourth decades). Excluding leukemias and malignant lymphomas, testicular tumors constitute the leading form of lethal cancer