In vitro studies of gonadal organogenesis in the presence and absence of H-Y antigen

Summary In a very strict sense, the primary (gonadal) sex of mammals is determined not so much by the presence or absence of the Y but the expression or nonexpression of the evolutionary extremely conserved plasma membrane H-Y antigen. The central somatic blastema of embryonic indifferent gonads contains one cell lineage characterized by the possession of S−F differentiation antigen that differentiates into testicular Sertoli cells in the presence of H-Y and into ovarian follicular (granulosa) cells in its absence. This cell lineage appears to play the most critical role in gonadal differentiation. Whether or not testicular Leydig cells and ovarian theca cells are similarly derived from the common cell lineage has not been determined. Nevertheless, if given H-Y antigen, presumptive theca-cell precursors of the fetal ovary acquire hCG (LH?)-receptors—the characteristic of fetal Leydig cells. Presented in the formal symposium on Sexual Differentiation in Vitro and in Vivo at the 29th Annual Meeting of the Tissue Culture Association, Denver, Colorado, June 4–8, 1978. This work was supported by Contract NO1-CB-33907, and Grants No. 1 RO1 AG00042 and No. 5 RO1 CA16952 from the National Institutes of Health.     

XY gonadal dysgenesis and the H-Y antigen

Summary H-Y antigen was determined in 12 patients affected by XY gonadal dysgenesis. Of these, three proved to be H-Y negative, and nine, including two sisters, were H-Y positive; two of the unrelated positive cases exhibited a reduced antigen titer. Therefore, this clinical condition must be genetically heterogeneous. It is assumed that in the negative cases and possibly in those with reduced antigen titer, the H-Y generating system is affected by mutation, while in the regular positive cases the target cells are unable to respond due to a defect of the gonad-specific H-Y antigen receptor.I dedicate this article to the memory of Ilse Aschmoneit     

H-Y antigen in 46,XY gonadal dysgenesis

Summary Presence of H-Y antigen has been correlated with testicular differentiation, and absence of H-Y with failure of testicular differentiation, in a variety of mammalian species. To determine more precisely the relationship between expression of H-Y antigen and development of the testis, we studied the cells of phenotypic females with the 46,XY male karyotype. Blood leukocytes were typed H-Y⁺ in five XY females with gonadal dysgenesis, although in other studies blood leukocytes from XY females with gonadal dysgenesis were typed H-Y^-. Thus mere presence of H-Y antigen is not sufficient to guarantee normal differentiation of the testis. In the present paper we review evidence for an additional factor in gonadal organogenesis, the H-Y antigen receptor. We infer that testicular development requires engagement of H-Y and its receptor. It follows that XY gonadal dysgenesis is the consequence of functional absence of the H-Y testis inducer as in the following conditions: failure of synthesis of H-Y or failure of specific binding of H-Y.     

H-Y antigen in Swyer syndrome and the genetics of XY gonadal dysgenesis

Summary The H-Y antigen is a plasma membrane antigen involved in the organogenesis of the mammalian testis. Its expression on human cells is determined by a Y-linked gene. Phenotypic females affected by 46,XY gonadal dysgenesis (Swyer's syndrome) can be either H-Y-positive or H-Y-negative. In this paper we report H-Y antigen and endocrine studies in a sibship with three affected sisters. Immunological studies were performed on two of the patients, and a clearly positive expression was detected in both cases. Endocrine studies consisted in the investigation of the hypothalamic-pituitary-gonadal axis, which revealed that gonadal hormone insufficiency is the only endocrine abnormality associated with the syndrome. A new genetic interpretation and classification of XY gonadal dysgenesis is proposed.     

H-Y antigen expression in a case of mixed gonadal dysgenesis

Summary H-Y antigen expression was studied on leukocytes and gonad-derived fibroblasts from a patient affected by mixed gonadal dysgenesis. Blood leukocytes and fibroblasts derived from the testis were typed H-Y positive, but the fibroblasts derived from the streak gonad were H-Y negative. Although the patient's karyotype was a mosaic, 45,XO/46,X+mar, as detected in-peripheral blood cells and testis-derived fibroblasts, all the fibroblasts derived from the streak gonad were 45,XO. These data suggests that the marker chromosome was in fact a Y-derived chromosome. Moreover, they showed that, at the gonadal level, a minority of H-Y positive 46,X+mar cells were able to organize a testis. Nevertheless, a large number of XO cells probably did not receive the testicular forming influence of the H-Y antigen and of the other masculinizing factors.     

Serological H-Y antigen in the female chicken occurs during gonadal differentiation

In the chicken, serological H-Y antigen is specific for the female sex. Male gonad differentiation can be experimentally influenced by estrogens, resulting in the transient formation of an ovotestis. The sex-inverted gonad becomes positive for H-Y antigen. Therefore, the question arises whether, in normal gonadogenesis also, the female gonad at the indifferent stage, before estrogens are produced, is negative for H-Y antigen. Here we show that this is indeed the case. The female gonad becomes positive for H-Y antigen when the ovary starts its organotypic differentiation at about day 6 1/2 of embryonal development. It is assumed that estrogens are responsible for the occurrence of H-Y antigen. This finding supports the view that H-Y antigen plays a role in primary ovogenesis in the chicken.     

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 vehicle-treated 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.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding studies of H-Y antigen in rat tissues

Summary 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.     

Evidence for a gonad-specific receptor for H-Y antigen: binding of exogenous H-Y antigen to gonadal cells is independent of beta 2-microglobulin.

This report addresses the question whether two different types of binding exist for the reaction of H-Y antigen with the cell surface. Anti-H-Y antiserum in the presence of complement was cytotoxic only for gonadal cells expressing their own H-Y antigen, but not to ovarian cells loaded with H-Y antigen. H-Y antigen was co-redistributed with beta 2--microglobulin on newborn testicular cells, but some residual H-Y activity was found on similarly treated testis cells from 15 day old rats. After beta 2--microglobulin redistribution, testis cells maintained their binding capacity for exogenous H-Y antigen prepared from epididymal fluid or Daudi cell culture supernatants. This result suggests that exogenous H-Y antigen is bound via a gonad-specific receptor which is independent of beta 2--microglobulin and that this type of binding for H-Y antigen is different from the beta 2--m-associated expression of H-Y antigen on the cell surface.     

In vitro regeneration in Primula ssp. via organogenesis

Culturing pedicle segments of primroses on a medium supplemented with 2,4-dichlorophenoxyacetic acid (2, 4-D) and thidiazuron (TDZ) resulted in callus induction rates of about 80%. The highest shoot regeneration rate (1.8 shoots per explant; mean of ten genotypes) was achieved with the combination of 2.0 mg/l 2, 4-D and 2.0 mg/l TDZ. Culture on a medium containing a high concentration of nitrate (for example, B5 medium) negatively affected the survival of regenerated shoots of one genotype, Gelb IV 48, probably due to an increase in the pH value of the medium. Consequently, the highest efficiency was obtained using a basal medium containing half-strength Murashige and Skoog macroelements. A protocol to regenerate shoots of Primula vulgaris and P. elatior is described.     

H-Y antigen: localization of the H-Y gene

Testicular development in a patient with deletion of the distal (fluorescent) segment of the Y chromosome is described. The presence of a normal dose of H-Y antigen was demonstrated by Goldberg's cytotoxicity test. It is concluded that the distal fluorescent segment of the Y chromosome is void of genes regulating H-Y antigen activity.     

H-Y antigen in the teleost

H-Y antigen, presumably the product of mammalian testis-determining genes, has been detected in three species of teleost fish, Xiphophorus maculatus. Haplochromis burtoni, and Oryzias latipes, and in hybrids of the genus Tilapia. In X. maculatus H-Y was most readily detected in YY males, suggesting that a genetic determinant of H-Y antigen expression may exist on the teleost Y-chromosome. Although H-Y was detected in males and not in feamles in each of the species that we studied, male heterogamety has not been firmly established for H. burtoni. Thus despite the extreme phlyogenetic conservation of H-Y genes and their association with the Y-chromosome, it remains open to question whether H-Y will always be found in the heterogametic sex, and whether serologically defined H-Y antigen plays any part in the differentiation of the teleost gonad.     

In vitro diameter response of rat femoral artery to flow in the presence and absence of endothelium.

We have examined the diameter response of rat femoral artery segments in the presence and absence of endothelium to changes in flow rate. The segments were isolated, mounted on microcannulae, maintained at 37 degrees C, and perfused at 90 mmHg with Tyrode's solution. The external arterial diameter was measured using video-microscopy. The mean control diameter was 741+/-22 microm (mean+/-SEM,n=7). The arteries were preconstricted to 75+/-1% of the control diameter with a superfusion of 1 microM norepinephrine (NE). Endothelial function was verified by perfusion of 1 micro;M acetylcholine (ACh). Two different flow protocols were employed: step changes in flow (n=7) and low-frequency sinusoidal flow changes (0.01Hz0.05). Sinusoidal flow oscillations resulted in sinusoidal diameter oscillations, whose amplitude and phase lag were inversely proportional to the frequency of the flow oscillations. A first-order low-pass filter, with a time constant of 28+/-3 and 30+/-5s for arteries with and without functional endothelium, respectively, was used to describe the relation between oscillatory flow and diameter. The response of the rat femoral arteries to changes in flow was not found to be different whether the endothelium was intact or removed.     

In vitro organogenesis and plant formation in cucumber

In vitro organogenesis was achieved from callus derived from hypocotyl explants of Cucumis sativus L. cv. Poinsett 76. Calli were induced from hypocotyl explants excised from 7-d-old seedlings grown on Murashige and Skoog (MS) medium containing 87.64 μM sucrose, 0.8 % agar, 3.62 μM 2,4-dichlorophenoxy acetic acid and 2.22 μM 6-benzyladenine (BA). Regeneration of adventitious buds from callus (25 shoots explant⁻¹) was achieved on MS medium supplemented with 8.88 μM BA, 2.5 μM zeatin and 10 % coconut water after two subcultures in the same medium at 30-d interval. Gibberellic acid (1.75 μM) favoured shoot elongation and indole 3-butyric acid (7.36 μM) induced rooting. Rooted plants were hardened and successfully established in soil.     

In vitro development of rat embryos undergoing organogenesis in heparin-plasma.

This study examines the use of heparin-plasma as a culture medium for mammalian postimplantation whole-embryo culture. The growth and differentiation of head-fold rat embryo explants over 48 hours in a standard serum medium was compared with development of same stage explants over 48 hours in a plasma medium prepared using sodium heparin. Heparin disrupted the morphological differentiation of embryos, in a concentration-dependent manner, from 25 micrograms sodium heparin/ml media (i.e., 5 IU/ml media), with overall embryo growth being adversely affected from a concentration of 200 micrograms sodium heparin/ml media (i.e., 40 IU/ml media). Defects of cranial neural tube development were the first apparent structural anomalies resulting from culture in heparin media. Forebrain development was grossly abnormal and associated with failure of eye development. As the heparin concentration in media increased, the cephalic neural folds remained widely open and the edges became increasingly everted, although differentiation of the heart, otic primordia, and pharyngeal arch persisted. Similar concentration-dependent dysmorphogenic effects were seen when embryos were cultured in the standard serum media with added heparin. A minimum heparin concentration of 100 micrograms sodium heparin/ml media (i.e., 20 IU/ml media) was required to effectively inhibit coagulation of the plasma medium over the 48 hour culture period. Although embryonic growth was not adversely affected at this heparin concentration, morphological differentiation was severely disrupted. Therefore, heparin is not a suitable anticoagulant for the preparation of plasma for use in postimplantation whole-embryo culture.     

In vitro effect of silica on axial organ cells in the presence and absence of mitogens

The axial organ (AO-cells) of the sea star Asterias rubens is a primitive immune organ. The total population was fractionated into two populations: adherent (B-like) and non-adherent (T-like) to nylon wool. Mitogenic responses were tested in the presence or absence of silica. Silica inhibits specifically the action of phagocytes contained in T-like and B-like subpopulations. Mitogenic responses do not occur in the presence of silica.     

In vitro organogenesis and plant formation in Acacia sinuata

In vitro morphogenesis via organogenesis was achieved from callus cultures derived from hypocotyl explants of Acacia sinuata on MS (Murashige and Skoog, 1962) medium. Calli were induced from hypocotyl explants excised from 7-day-old seedlings on MS medium containing 3% sucrose, 0.8% agar, 6.78 μM 2,4-dichlorophenoxyacetic acid and 2.22 μM 6-benzylaminopurine. Regeneration of adventitious buds from callus was achieved when they were cultured on MS medium supplemented with 10% coconut water, 13.2 μM 6-benzylaminopurine and 3.42 μM indoleacetic acid. Addition of gibberellic acid (1.73 μM) favored shoot elongation. Regenerated shoots produced prominent roots when transferred to half strength MS medium supplemented with 7.36 μM indolebutyric acid. Rooted plantlets, thus developed were hardened and successfully established in the soil. This protocol yielded an average of 20 plants per hypocotyl explant over a period of 4 months. This revised version was published online in June 2006 with corrections to the Cover Date.