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.     

Adenohypophysis regulates cell proliferation in the gonads of the developing chick embryo

The aim of the present study was to evaluate the effect of hypophysectomy on cell proliferation in the left ovary and the left testis of 8- to 14-day-old chick embryos. Hypophysectomy was performed by the partial decapitation technique. At 44-46 h of incubation, chick embryo heads were sectioned at the mesencephalic level and the prosencephalic region removed. Embryos were further incubated until 8-14 days of development. Cell division was evaluated by bromodeoxyuridine (BrdU) incorporation and by counting the total number of somatic and germ cells in the gonads. The ovary displayed an exponential increase in the number of somatic and germ cells and a higher rate of BrdU incorporation compared to the testis. BrdU incorporation was reduced in the ovary of hypophysectomized embryos at 9-14 days of incubation, while in the testis, the reduction was significant at 14 days of development. Changes in the total number of somatic and germ cells further suggest that the absence of hypophysis affects the growth of the ovary earlier than the growth of the testis. Reduction in the number of somatic and germ cells after hypophysectomy in the ovary was reversed by a hypophyseal graft on the chorioallantoic membrane. The adenohypophysis regulates, probably through gonadotropic hormones, proliferation of somatic and germ cells in the gonads during chick embryo development.     

Effects of steroid sex hormones on chick embryo gonads in organ culture, with special reference to hormonal control of gonadal sex differentiation

At the initial stages of sex differentiation (7.5 and 8.5 days of incubation), chick embryo gonads were treated directly with testosterone or estradiol-17 beta in organ cultures. Chemically-defined media containing cholesterol as a steroid precursor were used. The differentiation of gonads in the 10 to 12-day controls, cultured in media containing no hormones, was close to that of gonads of equivalent age in ovo. Testosterone added to the medium exerted an inhibitory effect on the cortex of the female gonad and a masculinizing one on its medulla. The results of estradiol treatment confirmed the known feminizing effect of that hormone on the male gonad, the meiotic prophase in the genetically male germ cells being initiated in the induced cortex. These data may be interpreted in favour of a bihormonal theory of gonadal sex differentiation in birds, where the predominantly-synthesized male or female hormone in the gonad determines the male or female pattern of development of the corresponding gonad.     

Transfer of blood containing primordial germ cells between chicken eggs development of embryonic reproductive tract

The present study was carried out to investigate development of recipient chicken embryonic reproductive tracts which are transferred chicken primordial germ cells (PGCs). It is thought that differentiation of PGCs is affected by the gonadal somatic cells. When female PGCs are transferred to male embryos, it is possible that they differentiate to W-spermatogonia. However, the relationship development between PGCs and gonads has not been investigated. At stage 12–15 of incubation of fertilized eggs, donor PGCs, which were taken from the blood vessels of donor embryos, were injected into the blood vessels of recipient embryos. The gonads were removed from embryos that died after 16 days of incubation and from newly hatched chickens and organs were examined for morphological and histological features. The survival rate of the treated embryos was 13.6% for homo-sexual transfer of PGCs (male PGCs to male embryo or female PGCs to female embryo) and 28.9% for hetero-sexual transfer PGCs (male PGCs to female embryo or female PGCs to male embryo) when determined at 15 days of incubation. The gonads of embryos arising from homo-sexual transfer appeared to develop normally. In contrast, embryos derived from hetero-sexual transfer of PGCs had abnormal gonads as assessed by histological observation. These results suggest that hetero-sexual transfer of PGCs may influence gonadal development early-stage embryos.     

Responses to androgen in lines of chickens selected for mating behavior

Two experiments were designed to study the effects of and relationships between caponization and androgen therapy on the mating behavior of lines of chickens that have undergone bidirectional selection for number of completed matings. Males from the high (HM) and low (LM) selected lines and the Athens-Canadiań (AC) randombred control population were randomly assigned to groups. One-half was caponized and the other half was used as intact controls. Groups were administered 0 and 0.9 mg/day of testosterone propionate (TP) in.25 ml cottonseed oil for 14 consecutive days in the first experiment. In the second experiment the equivalent of 14 daily doses of 0, 0.4, 0.8, 1.6, and 3.2 mg/day of testosterone cypionate (TC) in cottonseed oil was injected as a single dose. Mating behavior was measured by placing individuals singly for 10 min into pens of females for 6 days and recording the number of courts, mounts, treads, and completed matings.     

Differences between male and female protogonia in chick embryos before sex differentiation of the gonads

The sex of chick embryos is diagnosed by cyto-karyological methods on skin fragments of 2-7 days' incubation before gonadal sex differentiation. In 44 males and 42 females statistical analyses have been made of the number and dimension of the germ cells, and of the volume of the gonadal primordia. Moreover an ultrastructural study has been made on the germ cells colonizing the genital ridges (70-hours of incubation). Early differences between the sexes have been found regarding: earlier numerical increase of CGs in the left gonadal primordium of the females; larger primordial germ cells in the female; the same cytological characteristics at ultrastructural level.     

Membrane-bound steel factor maintains a high local concentration for mouse primordial germ cell motility, and defines the region of their migration

Steel factor, the protein product of the Steel locus in the mouse, is a multifunctional signal for the primordial germ cell population. We have shown previously that its expression accompanies the germ cells during migration to the gonads, forming a "travelling niche" that controls their survival, motility, and proliferation. Here we show that these functions are distributed between the alternatively spliced membrane-bound and soluble forms of Steel factor. The germ cells normally migrate as individuals from E7.5 to E11.5, when they aggregate together in the embryonic gonads. Movie analysis of Steel-dickie mutant embryos, which make only the soluble form, at E7.5, showed that the germ cells fail to migrate normally, and undergo "premature aggregation" in the base of the allantois. Survival and directionality of movement is not affected. Addition of excess soluble Steel factor to Steel-dickie embryos rescued germ cell motility, and addition of Steel factor to germ cells in vitro showed that a fourfold higher dose was required to increase motility, compared to survival. These data show that soluble Steel factor is sufficient for germ cell survival, and suggest that the membrane-bound form provides a higher local concentration of Steel factor that controls the balance between germ cell motility and aggregation. This hypothesis was tested by addition of excess soluble Steel factor to slice cultures of E11.5 embryos, when migration usually ceases, and the germ cells aggregate. This reversed the aggregation process, and caused increased motility of the germ cells. We conclude that the two forms of Steel factor control different aspects of germ cell behavior, and that membrane-bound Steel factor controls germ cell motility within a "motility niche" that moves through the embryo with the germ cells. Escape from this niche causes cessation of motility and death by apoptosis of the ectopic germ cells.     

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.     

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.     

The control of the differentiation of female embryonic germ cells in the bird

The left gonad from female chick embryos at 4–12 days of incubation was cultured in vitro as pieces of intact gonad, pieces of isolated cortex, and groups of pure germ cells. All cultures were maintained for a time equal to 17 days in ovo. At the end of the culture period, a cytological and quantitative study was made on the germ cells.The results show that some germ cells in pieces of intact 6-day gonad and pieces of 6-day cortex complete their normal developmental sequence and enter zygotene. This shows that the factors that control the differentiation of the germ cells reside in the cortex of the gonad and their expression does not depend upon the pituitary and the medullary estrogens after 6 days of incubation.Germ cells that are cultured as isolated cells do not attach to the tissue culture substrate, do not divide mitotically, and do not enter zygotene. Evidence is presented that suggests 12-day germ cells do enter zygotene when cultured with pieces of 12-day cortex. These data suggest the differentiation of the female germ cells is regulated by the somatic cells of the cortex.     

Immunomagnetic purification of viable primordial germ cells of Japanese quail (Coturnix japonica).

Immunomagnetic cell sorting (MACS) with the monoclonal antibody (mAb) QCR1 was compared with the Ficoll density-gradient centrifugation system (FICS) in terms of the efficiency of enrichment of quail (Coturnix japonica) primordial germ cells (PGCs) from blood. The purified PGCs were tested for their ability to settle in the chick (Gallus domesticus) embryonic gonad. Blood containing 60-100 PGCs microliter-1 was taken from the dorsal aorta of quail embryos at Hamburger and Hamilton's stages 14-16. The amount and concentration of PGCs in the PGC-rich fraction purified by MACS were greater than in the fraction purified by FICS. Purified quail PGCs were transfused into chick embryos at stages 14-16 and immunohistochemically stained with mAb QCRI on day 8 of chick development. Transfused PGCs purified by either MACS or FICS were positively stained in the chick embryonic gonads.     

Improvement of transfection efficiency in cultured chicken primordial germ cells by percoll density gradient centrifugation

Chicken primordial germ cells (PGCs) differentiate into germ cells in gonads. Because PGCs can be cloned and cultured maintaining germline competency, they are a good means of modifying the chicken genome, but the efficiency of plasmid transfection into PGCs is very low. In this study, I attempted to improve the efficiency of PGC transfection. Cultured PGCs were purified by Percoll density gradient centrifugation, and were then transfected with plasmid DNA. For transient transfection, the transfection efficiency increased more than 7-fold by the Percoll method. The efficiency of stable transfection of PGCs also increased significantly. The stable transfectants that were isolated by this method accumulated in the developing gonads after microinjection into bloodstream of chick embryos, indicating that gene transfection by Percoll purification did not alter the function of PGCs in vivo.     

Germ cell cycles during sex inversion in chick embryos]

A study was made of the germ cell cycles of 11 days old embryos injected male or female hormones on the 4 th day of incubation. The cell cycles duration in genetically male 11 days embryos treated with esradiol-benzoate was close to that registered in oogonia of both treated and non-treated female embryos of the same age. The testosterone propionate injection caused an acceleration of the genetically male sex cell proliferation and a decrease of the reproduction rate of the female sex cells. It is proposed that under normal conditions female sex hormones inhibit a hypothethic factor that determines the decrease of cell proliferation during the male embryo development.     

Histochemical demonstration of cholesterol in differentiating chick gonads

Summary Histochemical reactions for lipids (Sudan Black) and for cholesterol (digitonine precipitation) were applied to gonads of chick embryos between seven and twelve days of incubation.Both reactions were negative in seven day gonads, being positive in ovaries of eight days or more. They were localized in ovarian medulla. Interstitial tissue of testes stained positively with Sudan after the eighth day, while cholesterol did not appear in it until the tenth incubation day.The positivity of these reactions at ages in which ovarian differentiation takes place, and in which the existence of feminizing substances has been demonstrated by other authors, gives support to the belief that estrogens are the inducers of ovarian differentiation.The possible meaning of negative results obtained in eight and nine day testes has been discussed.This work was supported with a grant of the Consejo Nacional de Investigaciones Cientificas y Técnicas of Argentina. Dr. Sabatini worked under a fellowship of the same Institution.     

Loss of PGC-specific expression of the orphan nuclear receptor ERR-beta results in reduction of germ cell number in mouse embryos

Estrogen related receptor beta (ERR-beta) is an orphan nuclear receptor specifically expressed in a subset of extra-embryonic ectoderm of post-implantation embryos. ERR-beta is essential for placental development since the ERR-beta null mutants die at 10.5dpc due to the placenta abnormality. Here, we show that the ERR-beta is specifically expressed in primordial germ cells (PGC), obviously another important cell type for reproduction. Expression of the ERR-beta mRNA in embryonic germ cells started at E11.5 as soon as PGC reached genital ridges, and persisted until E15-E16 in both sexes. Immunostaining with anti-ERR-beta antibody revealed that the ERR-beta protein is exclusively expressed in germ cells in both male and female gonads from E11.5 to E16. 5. To study function of the ERR-beta in PGC, we complemented placental defects of the ERR-beta null mutants with wild-type tetraploid embryos, and analyzed germ cell development in the rescued embryos. It was found that development of gonad and PGC was not apparently affected, but number of germ cells was significantly reduced in male and female gonads, suggesting that the ERR-beta appears to be involved in proliferation of gonadal germ cells. The rescued embryos could develop to term and grow up to adulthood. The rescued ERR-beta null male were found to be fertile, but both male and female null mutants exhibited behavioural abnormalities, implying that the ERR-beta plays important roles in wider biological processes than previously thought.     

Improvement of Transfection Efficiency in Cultured Chicken Primordial Germ Cells by Percoll Density Gradient Centrifugation

Chicken primordial germ cells (PGCs) differentiate into germ cells in gonads. Because PGCs can be cloned and cultured maintaining germline competency, they are a good means of modifing the chicken genome, but the efficiency of plasmid transfection into PGCs is very low. In this study, I attempted to improve the efficiency of PGC transfection. Cultured PGCs were purified by Percoll density gradient centrifugation, and were then transfected with plasmid DNA. For transient transfection, the transfection efficiency increased more than 7-fold by the Percoll method. The efficiency of stable transfection of PGCs also increased significantly. The stable transfectants that were isolated by this method accumulated in the developing gonads after microinjection into bloodstream of chick embryos, indicating that gene transfection by Percoll purification did not alter the function of PGCs in vivo.     

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.     

PITX2 controls asymmetric gonadal development in both sexes of the chick and can rescue the degeneration of the right ovary

The gonads arise on the ventromedial surface of each mesonephros. In most birds, female gonadal development is unusual in that only the left ovary becomes functional, whereas that on the right degenerates during embryogenesis. Males develop a pair of equally functional testes. We show that the chick gonads already have distinct morphological and molecular left-right (L-R) characteristics in both sexes at indifferent (genital ridge) stages and that these persist, becoming more elaborate during sex determination and differentiation, but have no consequences for testis differentiation. We find that these L-R differences depend on the L-R asymmetry pathway that controls the situs of organs such as the heart and gut. Moreover, a key determinant of this, Pitx2, is expressed asymmetrically, such that it is found only in the left gonad in both sexes from the start of their development. Misexpression of Pitx2 on the right side before and during gonadogenesis is sufficient to transform the right gonad into a left-like gonad. In ZW embryos, this transformation rescues the degenerative fate of the right ovary, allowing for the differentiation of left-like cortex containing meiotic germ cells. There is therefore a mechanism in females that actively promotes the underlying L-R asymmetry initiated by Pitx2 and the degeneration of the right gonad, and a mechanism in males that allows it to be ignored or overridden.     

A method to obtain avian germ-line chimaeras using isolated primordial germ cells.

Primordial germ cells (PGCs), collected from the blood of 2-day-old chick embryos, were concentrated by Ficoll density centrifugation. The blood contained 0.048% PGCs and the concentrated fraction contained 3.9% PGCs in blood cells. The PGCs were picked up with a fine glass pipette, and one hundred were then injected into the terminal sinuses of 2-day-old Japanese quail embryos (24 somites); bubbles were then inserted to prevent haemorrhage. The embryos were further incubated at 38 degrees C for 24 h, and then fixed. Serial sections were stained with the periodic acid-Schiff reagent (PAS) to demonstrate chicken PGCs and with Feulgen stain to identify quail cells. On the basis of the differences in staining properties, 63.6 +/- 5.3 chick PGCs were detected in the quail embryo in the area where the gonads develop. Furthermore, 39.3 +/- 4.5 chick PGCs were incorporated into the quail germinal epithelium within 24 h of the injection. A similar percentage of the host (quail) PGCs had also migrated to the germinal epithelium at the same stage of development. This technique for obtaining germ-line chimaeras will facilitate research on avian germ-line differentiation.     

Development of 7--11-week-old human embryonic gonads in organ culture]

The author suggests a simplified method of organic culture of the human gonads. Gonads (160) from 7--11-week embryos were cultivated on millipore filters in penicillin bottles. During the first week of cultivation the central necroses were observed both in the male and the female gonads, but later they disappeared in some of the explants. In the ovaries from the 9--11-week embryos the germ cells underwent development which continued to the pachytene stage, while in the ovaries from the 7--9-week embryos the development of the germ cells failed to reach the leptotene stage. In the case of testis germ cells they remained at the stage of spermatogonia.