Tamoxifen and sex differentiation of the gonads in chick embryo

Tamoxifen or 4-hydroxytamoxifen were injected either alone or in combination with oestradiol into 4-5 day-old chick embryos in order to study their action on the sex differentiation of the gonads. The results of the histological study of the gonads performed at the stage of 16-19 days warrant the following conclusions: None of both anti-oestrogens exerts an effect on the testes. None of both compounds modifies the sex differentiation of the female gonads. Tamoxifen exerts an antagonistic action on the feminization of the testes by oestradiol. These conclusions do not lend support to the hypothesis according to which oestrogens play a role in normal sex differentiation of the female gonads.     

Interaction of the anti-oestrogen, nafoxidine hydrochloride, with the soluble nuclear oestradiol-binding protein in chick liver.

Nafoxidine hydrochloride (Upjohn, 11100A)injected with oestradiol into immature chicks inhibits the hormone-induced increase in [3H]oestradiol-binding activity in salt extracts of liver nuclei as well as the subsequent production by liver of egg-yolk phosphoprotein. Substantial inhibition of both oestradiol-induced responses is seen when nafoxidine is given in a dose approximately equimolar with that of oestradiol. In vitro nafoxidine competitively inhibits binding of [3H]oestradiol in nuclear extracts. The Ki for the inhibition is 43 nM, which indicates an affinity of nafoxidine for the binding protein about 4% of that of oestradiol. The inhibitory action of nafoxidine in vivo thus is more potent than the relative binding affinity determined in vitro might indicate. One possible explanation is that the primary site of nafoxidine action is at a point proximal to nuclear receptor interaction. Nafoxidine injected alone into the chick does not induce phosphoprotein synthesis, but it does increase [3H]oestradiol-binding activity in extracts of liver nuclei to a limited extent. No differences in the properties of the oestradiol-binding activity in extracts from nafoxidine-treated chicks or from oestradiol-treated chicks were detected. Chick liver cytosol does not contain detectable high-affinity oestradiol-binding activity. A low-affinity oestradiol-binding component with a sedimentation coefficient of 3.5S was found, but it was unaffected by treatment of chicks with earlier nafoxidine or oestradiol. The results suggest a difference in the mechanism of oestradiol action in the chick liver and in the widely studied rat uterus, on which the usual model for oestradiol action is largely based.     

Effect of LH on progesterone and oestradiol production in vivo and in vitro by preovulatory rat follicles

Temporal changes in follicular oestradiol production induced in vitro and in vivo by LH were studied. In-vitro changes were measured by incubating preovulatory rat follicles for 12 h, changing the medium every 2 h. Follicles isolated at various intervals after an injection of 10 i.u. hCG were incubated for 2 h to measure changes in oestradiol production in vivo. In both studies there was an increase in oestradiol production lasting 4 h followed by a sharp decline. Progesterone production was also increased by LH in vitro or hCG in vivo, but remained high. A second exposure to LH did not raise oestradiol synthesis, but increased progesterone synthesis in vitro only. The decline in oestradiol production is most probably due to a decrease in C17-20 lyase activity, because addition of testosterone, but not of 17 alpha-hydroxyprogesterone, increased oestradiol production. Incubation of preovulatory follicles in the absence of LH or incubation of follicles derived from animals in which the spontaneous LH surge was blocked by an injection of pentobarbitone sodium also resulted in a decrease of oestradiol and an increase in progesterone production. This oestrogen-progesterone shift was also caused by a decrease in C17-20 lyase activity. The results demonstrate that the changes in steroid production in vivo and in vitro are similar and occur in the presence and absence of LH. It is concluded that the decrease in oestradiol production is dependent on the decrease in the activity of enzymes converting progesterone to aromatizable androgens.     

The overlapping effects of thyrotropin and gonadotropins on chick embryo gonads in vitro

Pieces of 12- and 15-day-old chick embryo testes and ovaries were cultured in vitro in the presence of thyrotropin (TSH), gonadotropins (FSH + LH) and adrenocorticotropin (ACTH) for different periods. All the explants of treated gonads differentiated into typical testes or ovaries according to their genetic sex. The gonads of 12-and 15-day-old chick embryos showed a good response to both thyrotropic and gonadotropic stimulation. On the other hand, they did not respond to adrenocorticotropic stimulation. Fifteen-day-old chick embryo testes were grown in tissue culture in the presence of the said hormones. Gonadotropins and TSH enhanced the growth and migration of testicular cells as compared with the control or ACTH treated group. In addition, they maintained the germ cells on the upper surface of epithelial cells. These results have confirmed our previous results in vivo in that gonadotropins and thyrotropin hormones accelerated the development of 12- or 15-day-old chick embryo gonads.     

Effects of in vitro culture of mouse fetal gonads on subsequent ovarian development in vivo and oocyte maturation in vitro

Under organ culture, female fetal gonads in mice cannot develop beyond the preantral follicle stage unless the follicles are individually isolated and cultured again. In this study, we investigated the effect of in vitro culture of female fetal gonads before transplantation on subsequent in vivo development. The gonads derived from female fetuses 12.5 days postcoitum were organ-cultured for 0, 7 and 14 days, and then were grafted underneath the kidney capsules of severe combined immunodeficient mice and recovered at 21, 14 and 7 days post-transplantation, respectively. The histological analysis of the grafts showed that the in vitro culture of the fetal gonads restricted follicular development to the antral follicle stage post-transplantation. In the grafts cultured for 14 days, particularly, no antral follicle was observed. However, the oocytes in these follicles had grown to around 65 µm in diameter and had competence to resume meiosis in vitro . When the fetal gonads were grafted after culture for 7 and 14 days, 13.0% and 6.8% of the oocytes progressed to the metaphase II stage, respectively. These data showed significant differences ( P  < 0.05) in comparison with the control group (25.3%). Our results indicate that the in vitro culture of female fetal gonads before transplantation affects the subsequent in vivo development of both follicular cells and oocytes, and in vitro oocyte maturation. However, this effect seems to be more severe in terms of follicular development when compared with oocyte growth and maturation.     

Evidence for a role for anti-Mullerian hormone in the suppression of follicle activation in mouse ovaries and bovine ovarian cortex grafted beneath the chick chorioallantoic membrane

The first critical transition in follicular development, the activation of primordial follicles to leave the pool of resting follicles and begin growth, is poorly understood, but it appears that the balance between inhibitory and stimulatory factors is important in regulating the exodus of follicles from the resting pool. There is evidence that anti-Mullerian hormone (AMH; also known as MIS) inhibits follicle activation in mice, but whether it plays a similar role in non rodent species is not known. When pieces of bovine ovarian cortex, rich in primordial follicles, are cultured in serum-free medium, most follicles initiate growth, but when cortical pieces are grafted beneath the chorioallantoic membrane (CAM) of chick embryos, follicle activation does not occur. Since embryonic chick gonads of both sexes produce and secrete high levels of AMH, the hypothesis that the AMH in the chick circulation inhibits follicle activation was tested. In Experiment 1, whole newborn mouse ovaries were grafted beneath the CAM (placed "in ovo") or cultured in vitro for 8 days. In vitro (or after 8 days in vivo) follicles activated and proceeded to the primary or secondary stage, but activation was suppressed in ovo. This inhibition was reversed if ovaries were removed from beneath the CAM and cultured in vitro. In contrast, when ovaries from mice null mutant for the AMH type II receptor were CAM-grafted in Experiment 2, follicle activation occurred in a similar fashion to activation in vitro. This finding strongly implicates AMH as the inhibitor of follicle activation in ovo. Since chick embryonic gonads are the source of circulating AMH, chicks were gonadectomized in Experiment 3, prior to grafting of pieces of bovine ovarian cortex beneath their CAMs. Bovine primordial follicles activated in the gonadectomized chicks, similar to the results for mice lacking the AMH type II receptor. Taken together these experiments provide strong evidence that AMH is the inhibitor of mouse follicle activation present in the circulation of embryonic chicks and provide indirect, and hence more tentative, evidence for AMH as an inhibitor of bovine follicle activation.     

Chick gonad differentiation following excision of primordial germ cells

The differentiation of embryonic chick gonads lacking germ cells was compared to that of normal chick gonads to determine whether the somatic elements of sterile avian gonads will undergo normal sexual differentiation. Primordial germ cells were removed by surgical excision of anterior germinal crescent from early embryos, Hamburger and Hamilton stages 6–11. Surgically treated and control embryos were sacrificed at 6, 15, and 20 days of incubation, and their gonads were studied histologically. Analysis of differentiation was based on morphological criteria at the cellular, tissue, and organ levels. In both male and female embryos, the somatic elements of the gonads differentiated normally in the absence of germ cells. The significance of these results for understanding the controls of differentiation of both the somatic gonad and the germ cells in birds is discussed and correlated with similar results in mammals.     

Sex Differentiation of Avian Gonads In Vitro

The analysis of avian sex differentiation in vitro has beenlimited to the following problems: morphological sex differentiationof gonads cultured in vitro; analysis of the chemical natureof the hormonal secretion; differentiation of germ cells inrelation to their somatic environment. Morphological sex differentiationof avian gonads occurs in vitro. Differentiated gonads of thechick embryo carry out biosynthesis of sex hormones from severalradioactive precursors. Female gonads in particular synthesizeestrogens while male gonads synthesize testosterone. Some experimentshave given evidence of estrogen synthesis by undifferentiatedfemale gonads. Embryonic gonads of quail, like those of chick,are able to synthesize sex steroids from radioactive precursors.However, in the quail and mainly in the testes, a delayed appearanceand a lower activity of the enzyme system 3ß-HSDHs-4-isomerase was found. Histoenzymological results corroboratethe biochemical ones. Combination of culture and grafting experimentshave shown that male germ cells when they are forced into femaledifferentiation by early colonization of a female gonad degenerateafter entering the premeiotic stage. The reasons for this delayedfailure of sex differentiation of "male oocytes" have certainlyto be searched for at the level of perturbation in the mechanismsof meiosis.     

Anti-fertility effects of embelin in female Sprague-Dawley rats may be due to suppression of ovarian function

Effects of embelin on oestrous cycle, plasma levels of progesterone and oestradiol, and in vitro production of oestradiol and progesterone by mixed ovarian cells was studied. Forty adult (4 months old) regularly cycling female Sprague-Dawley rats were divided into four groups of 10 rats each. Groups I and II (controls) were given 1 ml/kg body weight of physiological saline or corn oil (vehicle). Groups III and IV received 10 mg/kg and 20 mg/kg body weight embelin in corn oil, respectively. Emberlin disrupted the oestrous cycles in Groups III and IV animals, and there was a significant depression in plasma oestradiol (p <0.05) and progesterone (p <0.02) at both 10 and 20 mg/kg body weights, respectively. Isolated mixed ovarian cells from embelin treated rats produced significantly less progesterone and estradiol than controls in vitro. It is concluded that embelin probably interferes with reproductive functions in female rats by suppressing ovarian production of sex steroid hormones.     

Inhibition of thyroxine binding to adenohypophysial proteins in vitro by 2,4-dinitrophenol administered in vivo.

Male and female rats were given oestradiol benzoate (1 mg as a microcrystal aqueous suspension i.m. twice a week), 0.0033% 2.4-dinitrophenol (DNP) in their food (about 1 mg/rat/day), or 0.1% DNP in their food (about 30 mg/rat/day), or both oestradiol and DNP. The smaller DNP dose mildly stimulated food consumption and did not affect body weight. The larger dose strongly inhibited food consumption in the first two weeks of the experiment; consumption then returned to the control level, but body weight fell markedly at the same time. After 3 weeks' administration of both the small and the large dose of DNP, adrenal weight in the males was raised and the weight of the gonads was unchanged. The large DNP dose severely reduced the weight of the seminal vesicles and the uteri. It also inhibited the accumulation of radioiodine in the thyroid of both males and females. Isolated administration of the oestrogen raised adrenal weight in the males and ovarian and uterine weight in the females; it reduced the weight of the testes and seminal vesicles. These reactions were not affected by DNP. A pronounced oestradiol-induced increase in the weight of the adenohypophyses was accompanied by raised thyroxine binding to the adenohypophysial proteins in vitro. DNP inhibited the growth reaction of the adenohypophysis to the oestrogen only slightly and non-significantly, but significantly inhibited the thyroxine binding reaction to the adenohypophysial proteins in vitro. By itself, DNP had no effect on adenohypophysial weight, but reduced thyroxine binding to the adenohypophysial proteins in vitro, especially in males. The effect of DNP was similar to that of thyroxine observed in earlier experiments; nothing is known of its mechanism.     

Early Morphogenesis of Chick Gonad in the Absence of Mesonephros

The development of the gonads in male and female chick embryos with induced unilateral mesonephric agenesis was studied using grafting, histoenzymology, and electron microscopy. As in embryos with a mesonephros, proliferation of the coelomic epithelium and its interaction with mesenchymal cells to form the medullary cords take place in the amesonephric gonads. In a similar manner, gonadal sexual differentiation and the differentiation of steroidogenic tissue, detectable by the presence of Δ5-3β-hydroxysteroid dehydrogenase, do not appear to be affected by the absence of an organized mesonephros. However, the initiation of gonadal development, further growth, and the onset of meiosis observable in developing ovaries are retarded. This delay appears to be reversible, as was demonstrated by experiments in which ovaries from chicks with complete mesonephric agenesis were transplanted into the coelomic cavity of male and female 3 1/2-day-old embryos. Meiosis finally occurred in the oocytes of all ovaries, regardless of the sex of the host. Therefore, the presence of a differentiated mesonephros in chick embryos is not required for the establishment of an undifferentiated gonad and sexual differentiation, or for initiation of meiosis.     

Effect of pentobarbitone sodium and bromocriptine on follicular oestradiol production in the rat

Injection of an ovulation-blocking dose of pentobarbitone sodium given in the early afternoon of pro-oestrus in rats decreased follicular oestradiol production in vitro the next day (2.42 +/- 0.11 ng/4 h/follicle in pro-oestrous rats, 0.49 +/- 0.04 ng/4 h/follicle in pentobarbitone-treated rats). Pentobarbitone, given 1 day earlier (at dioestrus II), prevented the increase in oestradiol production that normally occurs between di-oestrus II and pro-oestrus. Injection of a subovulatory amount of hCG (0.5 i.u.) given after pentobarbitone injection inhibited the decrease in follicular oestradiol production induced by pentobarbitone. The pentobarbitone-induced decrease in oestradiol production was also prevented by bromocriptine (1 mg) given at di-oestrus II (15:00 h) and pro-oestrus (09:00 h). Bromocriptine is an effective inhibitor of prolactin secretion and this suggests therefore that the decrease in follicular oestradiol production after pentobarbitone is due to the preovulatory surge of prolactin. However, pretreatment with bromocriptine also inhibited the effect of pentobarbitone on oestradiol production when pentobarbitone was given at di-oestrus II. Moreover, when ergocornine (another inhibitor of prolactin secretion) was used instead of pentobarbitone to block ovulation, follicular oestradiol production was also decreased the next day. In contrast to bromocriptine, ergocornine was not able to prevent the pentobarbitone-induced decrease in follicular oestradiol production. These results indicate that the decrease in follicular oestradiol production after pentobarbitone injection is due to inhibition of the serum concentrations of LH rather than the preovulatory surge of prolactin. How bromocriptine (but not ergocornine) prevents the pentobarbitone-induced decrease in oestradiol production is not clear.     

Sex inversion in domestic chicken (Gallus gallus domesticus) by letrozole and tamoxifen

In multicellular organisms, determination of sex identity is a complex, multistage process. Sex hormones are synthesized in gonads and fulfill the role of inductors in this process. The effect of androgen is currently well studied. However, the participation of estrogen in the formation of female gonads and female sex on the whole is not much known. Here, we present the results of experimental sex inversion by inhibition of aromatase (an enzyme involved in estrogen synthesis) and tamoxifen (a modulator of estrogen receptors) in chick embryos. It was shown that masculinization depended on the dose of the substance and quantity of injections. Inhibition of aromatase did not block the meiotic prophase in oogoniums. It has been suggested that retinoic acid and estrogens have different mechanisms of effect on oogenesis. Proteins and nucleoproteins interacting with estrogen receptor 1 and their gene localization in human and chicken genomes have been shown for the first time.     

Crossed graft of embryonic testis between quail and chick embryos: contribution to the study of the mechanism of female bird embryo masculinization]

Quail, or chick embryonic testes grafted respectively in the extraembryonic coelom of chick or quail embryos induce both a Mullerian duct regression and a masculinization of the female host gonads up to the differentiation of two testes, in some cases. Such a result confirms the fact evidenced previously in other bird species (chick and duck) that the testis-inducer is interspecific. Quail cells are not observed in histological sections of embryonic gonads of testis-grafted chicks. This allows to discard a possible influence of cells migrating from the graft to the host. The grafted testes act then through substance/s secreted in the blood stream. Present and previous experimental data strongly suggest that the same substance, i.e. the so-called anti-Mullerian hormone, is responsible for both MD regression and gonadal sex reversal.     

The effect of prenatal stress on steroidogenesis in the gonads of Arctic foxes (Alopex lagopus)

Handling is a source of stress for farm-bred blue foxes. The influence of handling during the late gestational period was investigated in 10-day old male and female blue foxes. Testosterone and estradiol were measured by RIA in the plasma, gonadal homogenates and in vitro incubates from blue foxes of both sexes. The gonads were incubated in vitro without or with human chorionic gonadotropin. In cubs of both sexes, the gonad weights and ovarian estradiol production were decreased by stress. The testicular testosterone and ovarian estradiol contents were increased in prenatally stressed cubs as compared to the controls. The testicular content and baseline in vitro production of testosterone were not affected by prenatal stress, but the testicular response to human chorionic gonadotropin was higher in the stressed group. This study suggests that prenatal stress induced by handling pregnant vixens may influence gonadal steroidogenesis and this effect was more pronounced in female cubs.     

Reproductive endocrinology of the Black-browed albatross Diomedea melanophris and the Grey-headed albatross D. chrysostoma

Gonadal size and the circulating concentrations of two pituitary hormones (luteinizing hormone and prolactin) and three gonadal steroids (testosterone, progesterone and oestradiol-17β) were measured in two closely related Diomedea albatrosses at South Georgia. The Grey-headed albatross D. chrysostoma , if successful in rearing a chick, usually breeds biennially, whilst the Black-browed albatross D. melanophris normally breeds annually. Direct examination (by laparoscopy) of the gonads showed that the testes of both species underwent annual cycles, whilst endocrine data confirmed that those male Grey-headed albatrosses at the colony in the pre-laying period but not breeding in that year (having bred successfully the previous year) were apparently in full reproductive condition with elevated testosterone levels typical of breeding birds. However, the females of the two species differed markedly. Grey-headed albatrosses, in a year following successful breeding, had undeveloped ovaries with low levels of circulating oestradiol but high levels of progesterone, whereas the Black-browed albatrosses showed a pattern consistent with annual ovarian development. The profiles of gonadal steroids through the breeding season were similar for the males of both species but differences existed between the females. In the female Grey-headed albatrosses, transient peaks of progesterone were present throughout chick rearing but these were absent from Black-browed albatrosses. Prolactin had a similar profile in both species, with uniformly high levels throughout incubation and a rapid fall near the end of the brood-guard period. It is suggested that Grey-headed, like Black-browed, albatrosses are intrinsically annual breeders. However, if a female Grey-headed albatross breeds successfully in one year, then nutritional factors operate to ensure that in the following year the female does not show ovarian development, although the ovary is active in terms of progesterone secretion.     

Expression of fibronectin and laminin in fetal male gonads in vivo and in vitro with and without testicular morphogenesis

Sertoli cell differentiation occurs in vitro, even when testicular morphogenesis is inhibited by addition of serum to the culture medium (Magre, S. and A. Jost: Proc. Natl. Acad. Sci. USA 81, 7831-7834 (1984]. Using indirect immunohistochemical technique, we have studied the expression of fibronectin and laminin in gonads lacking testicular morphogenesis, as compared to in vivo controls and gonads cultured in synthetic medium. In undifferentiated gonads in vivo, fibronectin and laminin are distributed uniformly in the blastema. If testicular differentiation occurs in vivo, laminin is detected only in the basement membranes; when it occurs in vitro, laminin is found both in the basement membranes and among the stromal tissue. In gonads without seminiferous cords (cultured in serum-supplemented medium), fibronectin and laminin are both present, they are uniformly distributed among the gonadal cells.     

Germ line chimera produced by transfer of cultured chick primordial germ cells.

Intrinsic primordial germ cells (PGCs) from stage 27 (5-day-old) chick embryonic germinal ridges were cultured in vitro for a further 5 days, and shown to proliferate on stroma cells derived from the germinal ridge. To determine whether these cultured PGCs could colonize and contribute to the germ-line, PGCs were isolated by gentle pipetting, labeled with PKH26 fluorescent dye and injected into the blood stream of stage 17 (2.5-day-old) chick embryos. The recipient embryos were incubated until they reached stage 28. Thin sections of these embryos were analysed by fluorescent confocal laser microscopy. These analyses showed that the labeled donor PGCs had migrated into the germinal ridges of the recipient embryos, and transplanted PGCs had undergone at least 3-7 divisions. These results suggest that PGCs that had passed far beyond the migration stage in vivo were still able to migrate, colonize and proliferate in recipient chick embryonic gonads.     

Embryonic sex hormones in birds

Hormone activity of embryonic gonads in birds was demonstrated by grafting and culture experiments. Anti-Müllerian hormone responsible for the regression of the Müllerian ducts in the male is most probably a glycoprotein. Whether the testis also secretes testosterone has long been disputed, but most arguments are against this possibility. From early stages of development, the ovary secretes estrome and estradiol. However, it could not be demonstrated unambiguously whether estrogen is identical with the sex inducing substance in the female. The hypophysis seems to control ovarian estrogen secretion at 10-13 days of incubation in the chick embryo.     

Control of ovarian cholesterol ester biosynthesis

1. Experimental evidence is presented for a role of progesterone and 20alpha-hydroxypregn-4-en-3-one as inhibitors of cholesterol ester synthetase in the acute depletion of ovarian cholesterol ester after trophic stimulation. 2. Luteinizing hormone in vitro decreased by 84% the rate of esterification of cholesterol with added [(14)C]oleate by slices of rabbit ovarian interstitial tissue; this effect was mimicked by cyclic AMP (adenosine 3':5'-cyclic monophosphate) in vitro, and occurred without large changes in precursor pool sizes or membrane permeability. 3. Cyclic AMP was shown to have no direct effect on cholesterol ester synthetase or cholesterol esterase in cell-free extracts of rabbit ovarian interstitial tissue, but decreased the activity of cholesterol ester synthetase (not that of cholesterol esterase) in extracts prepared from slices previously incubated with it. 4. The inhibitory effect of cyclic AMP on esterification of cholesterol with added [(14)C]-oleate was prevented by both cycloheximide and aminoglutethimide phosphate (which also inhibited steroid synthesis in response to cyclic AMP). 5. Cyclic AMP raised the intracellular concentrations of progesterone and 20alpha-hydroxypregn-4-en-3-one in incubated slices by factors of 2.8 and 3.9 respectively. 6. Cycloheximide and aminoglutethimide phosphate administered in vivo blocked cholesterol ester depletion in response to luteinizing hormone in rats; in these ovaries cycloheximide and aminoglutethimide phosphate decreased the concentrations of progesterone and 20alpha-hydroxypregn-4-en-3-one and luteinizing hormone raised them. 7. Progesterone and 20alpha-hydroxypregn-4-en-3-one added to cell-free extracts of rabbit ovarian interstitial tissue in vitro (at concentrations comparable with those found in incubated slices) inhibited cholesterol ester synthetase by up to 85%. 8. The results are discussed with reference to the acute control of cholesterol ester concentrations in the ovary and adrenal cortex.