New insights on the mechanism of testis differentiation from the morphogenesis of experimentally induced testes in genetically female chick embryos

Embryonic testes grafted in the extraembryonic coelom of 3-day-old genetically female chick embryos may induce total and definitive reversal of gonadal sex differentiation. In this experimental condition, the left gonad becomes a testis instead of an ovary. This makes it possible to compare testicular and ovarian morphogenesis in animals having the same genetic sex and to discount what is due to differences in the genetic determination between male and female. The morphogenesis of such testes is marked by a disappearance of the cortical germinal epithelium. The medullary sex cords keep a narrow lumen instead of becoming large lacunae. The germ cells remain few in the sex cords and do not become meiotic. Furthermore, interstitial cell development is known to be very slow. As a consequence the gross size of the gonad is much smaller than that of an ovary. All these morphogenetic phenomena are unlike those observed during normal ovarian differentiation and evidence an inhibiting influence of the grafted testes. Since inhibition and masculinization are concomitant, inhibition appears to be the mechanism responsible for gonadal sex reversal. The extraembryonic situation of the grafted testes and their relation with the embryo only via the blood stream demonstrates the role of a secreted substance or substances still to be exactly identified. Previous data suggest that this could be the anti-Müllerian-hormone (AMH). Furthermore, previous and present results show that testis differentiation can be actively induced in a bird. This does not agree with the hypothesis that the gonads of the homogametic sex, i.e., the testes in birds, do not need any inducer in order to differentiate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of the testes in female domestic fowls submitted to an experimental sex reversal during embryonic life

The sex differentiation of the female chick embryo can be totally inverted toward the male sex by an early extraembryonic testis grafting. This sex reversal remains permanent, as shown by three adult fowls described in this paper. They possess two testes associated with normally differentiated male excretory ducts and their Müllerian ducts have regressed. The development of male sex characteristics such as external features, behavior and complete spermatogenesis is evidence that these cocks have endocrine and exocrine capabilities similar to those of normal cocks. Although these cocks were able to mate with female fowls, they were sterile. A mechanism is discussed by which grafted testes induce such modifications in females. Hypotheses considering the heterogametic sex (female in birds) as exerting a dominant influence on the phenotypic sexual differentiation can be discarded in light of our results because a homogametic testis provokes the definitive sex inversion of a female.     

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.     

Aminoglutethimide, an inhibitor of aromatase from chick embryo ovary

The gonads from 13-day-old female chick embryos were cultured in vitro on TC medium 199, and oestradiol production was measured by radioimmunoassay. In the presence of dehydroepiandrosterone as substrate, oestradiol synthesis was markedly increased, but when aminoglutethimide was also present, it was greatly reduced, depending on the concentration of the drug. This result demonstrates inhibition of the aromatizing enzyme system of the chick embryo ovary by aminoglutethimide in vitro. However, sex differentiation of the female gonads was not modified after in vivo treatment. Since it is not known whether their production is completely suppressed in vivo, the hypothesis cannot be dismissed that oestrogens play a role in ovarian differentiation.     

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.     

Synergistic action, on the masculinization of female chick embryo gonads, of a graft of embryonic testis and administration of an anti-estrogenic substance (tamoxifen)

The capability of an embryonic testis graft to induce a male gonadal sex differentiation in genetically female chick embryos was significantly enhanced by the administration of a antiestrogenic drug (tamoxifen). This observation reinforces the view that the experimentally-induced testis differentiation depends chiefly on a inhibition of estrogen influence.     

二化螟雄蛾交配行为与精巢大小的关系

【目的】为了明确二化螟Chilo suppressalis雄蛾交配行为与精巢大小的关系,阐明信息素群集诱杀防治二化螟的有效性。【方法】本研究通过测定和比较不同日龄、交配状态以及性信息素诱捕的二化螟雄蛾精巢体积,建立精巢体积与雄蛾发育及交配状态的相关性。【结果】雄蛾日龄显著影响二化螟的交配,刚羽化(0日龄)雄蛾的交配率较低,1日龄雄蛾的交配率最高,之后随着日龄增加交配率逐渐降低。精巢体积与二化螟雄蛾日龄之间存在显著的负相关性。同一日龄交配的二化螟雄蛾的精巢体积显著大于未交配雄蛾;性诱捕器诱捕雄蛾的精巢体积显著大于未被性诱捕器诱捕雄蛾,与已交配雄蛾的精巢体积相似。交配之后,精巢的发育过程和未交配雄蛾相似。田间性信息素引诱二化螟雄蛾精巢体积小于0-1日龄未交配雄蛾,大于2-6日龄未交配雄蛾。【结论】本研究结果表明,二化螟雄蛾对雌蛾性信息素的反应与其精巢大小存在正相关性;性信息素引诱的二化螟雄蛾具有更强的性信息素反应能力,同时性信息素引诱的大部分雄蛾为未交配状态;交配过程并不影响二化螟雄蛾精巢体积,也不影响精巢发育。二化螟交配能力与精巢大小的关系在理论上解释了性信息素群集诱杀二化螟的防控有效性,并为其他蛾类昆虫的交配研究提供依据。     

Studies of cytochrome P-450 in testis microsomes

Studies on the role of cytochrome P-450 in mouse, rat, and chick testis microsomes showed that this CO-binding hemoprotein is involved in the activity of the 17α-hydroxylase. A 70–80% inhibition by CO of the 17α-hydroxylase activity was detected in rat and chick testis microsomes. In the mouse testis, the level of the enzyme activity is ten times greater than that of the rat. This partly explains why an acceleration of NADPH oxidation by progesterone can be observed in mouse but not in rat testis microsomes. In rat testis microsomes, type I binding spectra of cytochrome P-450 was observed with pregnenolone, progesterone, 17-hydroxyprogesterone, androstenedione, and testosterone. The apparent K_s values for progesterone and 17-hydroxyprogesterone were 0.50 and 1.00 μm, respectively.When NADPH is used to measure cytochrome P-450 levels in rat testis microsomes, CO formation resulting from a stimulation in lipid peroxidation by phosphate or Fe²⁺ was sufficient to bind with 50% of the total amount of cytochrome P-450. Substitution of phosphate by Tris reduced the amount of lipid peroxidation to minimal levels. On a comparable basis, no CO formation was observed in avian testis microsomes.An increase in the testicular levels of cytochrome P-450 resulted upon the administration of HCG and cyclic-AMP to 1-day-old chicks. The lack of stimulation of the cytochrome P-450 levels by progesterone and pregnenolone suggest that the hormonal stimulation of the P-450 levels is not due to substrate induction.     

Action of the testosterone treated chicken testis on the regression of the Müllerian duct]

After administration of testosterone propionate to male chick embryos and chickens, their testis have an activity, on the retrogression of mullerian ducts, much more important than that observed in testis of normal subjects of the same age, activity measured by grafting testis fragments in undifferentiated female chick embryos. The results suggest that testosterone gives such an effect by inhibiting pituitary gonadotropins, peculiarly FSH.     

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.     

Inhibition of chick embryo hepatic uroporphyrinogen decarboxylase by components of xenobiotic-treated chick embryo hepatocytes in culture. II.

A variety of xenobiotics, viz., 3,3',4,4'-tetrachlorobiphenyl (TCBP), sodium phenobarbital (PB), 3,5-diethoxycarbonyl-2, 4,6-trimethylpyridine (OX-DDC), and nifedipine, cause a decrease in uroporphyrinogen decarboxylase (UROG-D) activity, accompanied by uroporphyrin accumulation, in chick embryo hepatocytes in culture. In this study the activity of 17-day-old chick embryo hepatic UROG-D was determined by measuring the conversion of pentacarboxylporphyrinogen I to coproporphyrinogen I, and it was shown that a UROG-D inhibitor, previously reported to accumulate in TCBP-treated and PB-treated chick embryo hepatocytes in culture, also accumulates in OX-DDC-treated and nifedipine-treated chick embryo hepatocytes in culture. It was concluded that the accumulation of a UROG-D inhibitor provides an explanation for the UROG-D inhibition observed in this culture system with xenobiotics that cause uroporphyrin accumulation. Studies of the UROG-D inhibitory fraction isolated from the 10,000 x g, 40,000 x g, and 100,000 x g supernatant fractions of cultured chick embryo hepatocyte homogenate led to the conclusion that the UROG-D inhibitor is derived from a soluble component of the homogenate.     

Effect of cyclic AMP on retrogression of the Mullerian ducts in chick embryos]

Theophyllin and puromycine, inhibitors of the enzyme phosphodiesterase and AMPc are all able to inhibit the retrogression of mullerian ducts in the female chick embryo, grafted with an embryonic testis. We can think that these results are explained by an inhibitory action of AMPc on the mechanisms responsible for the mullerian retrogression. So the chick embryo reacts similarly as do the mammalian embryo.     

Inhibiting effect of the testis on the ovary of the chick embryo]

The testicular hormone responsible for the retrogression of the mullerian ducts in the female chick embryo submitted to an embryonic testicular graft, causes an atrophy of the two, cortical and medullary, parts of this gonad, which is more marked at the level of the cortex.     

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.     

Feminization of the quail by early diethylstilbestrol treatment: histoenzymological investigations on steroid dehydrogenases in the gonads.

Early treatment of Quail eggs by DES promotes a transient feminization of the gonads in genetic males and a strong stimulation of the Müllerian ducts. The left ovotestis results from the juxtaposition of a testicular medulla and an induced female-type cortex, which develops follicles and a characteristic 17 beta-HSD activity. The right testis is reduced but keeps a consistent structure. The medulla of the treated gonads shows, in both sexes, an inhibition of delta 5-3 beta HSD activity during embryonic development. After hatching, this specific enzyme then develops in the steroidogenic cells. These results are compared with others obtained with estradiol and also in chick. The discussion deals also with the effects of these estrogens on the endogenous abilities and specific responses of the gonads in relation to sex differentiation factors.     

Influence of puromycin on the regression of Mullerian ducts in chick embryos submitted to a testicular graft]

Puromycin, an inhibitor of protein synthesis, strongly reduces the incidence of retrogression of the mullerian ducts provoked by testicular graft in the female chick embryo.     

Studies of gonadal sex differentiation

Gonadal differentiation has a determinative influence on sex development in human embryos. Disorders of sexual development (DSD) have been associated with persistent embryonal differentiation stages. Between 1998 and 2015, 139 female patients with various (DSD) underwent operations at the Scientific Center of Obstetrics, Gynaecology and Perynatology in Moscow, Russia. Clinical investigations included karyotyping, ultrasound imaging, hormonal measurement and investigations of gonadal morphology. The male characteristics in the embryo are imposed by testicular hormones. When these are absent or inactive, the fetus may be arrested at between developmental stages, or stay on indifferent stage and become phenotypically female. A systematic analysis of gonadal morphology in DSD patients and a literature review revealed some controversies and led us to formulate a new hypothesis about sex differentiation. Proliferation of the mesonephric system (tubules and corpuscles) in the gonads stimulates the masculinization of gonads to testis. Sustentacular Sertoli cells of the testes are derived from mesonephric excretory tubules, while interstitial Leydig cells are derived from the original mesenchyme of the mesonephros. According of the new hypothesis, the original mesonephric cells (tubules and corpuscles) potentially persist in the ovarian parenchyma. In female gonads, some mesonephric excretory tubules regress and lose the tubular structure, but form ovarian theca interna and externa, becoming analogous to the sustentacular Sertoli cells in the testis. The ovarian interstitial Leydig cells are derived from intertubal mesenchyme of the mesonephros, similar to what occurs in male gonads (testis). Surprisingly, the leading determinative factor in sexual differentiation of the gonads is the mesonephros, represented by the embryonic urinary system.     

Effects of Maternal Zinc Glycine on Mortality,Zinc Concentration,and Antioxidant Status in a Developing Embryo and 1-Day-Old Chick

This study was conducted to investigate the effects of maternal zinc glycine (Zn-Gly) supplementation as an alternative for zinc sulfate (ZnSO₄) on mortality, zinc (Zn) concentration, and antioxidant status in a developing embryo and 1-day-old chick. Six hundred 39-week-old broiler breeders were randomly assigned to 6 treatments, each treatment including 5 replicates with 20 birds each. Six treatments received a basal diet (control, 24 mg Zn/kg diet) or a basal diet supplemented with ZnSO₄ (80 mg Zn/kg) or Zn-Gly (20, 40, 60, or 80 mg Zn/kg), respectively. The experiment lasted for 8 weeks after a 4-week pre-experiment with a basal diet. At the last week, 100 eggs per replicate were randomly collected for incubation. Compared with the control treatment, Zn supplementation decreased (P < 0.05) embryo mortalities of the late stage and the whole period, increased (P < 0.05) liver Zn concentration in the embryo of d9, d19, and 1-day-old chick, and improved (P < 0.05) antioxidant status in the embryo of d19 and 1-day-old chick. Compared with the ZnSO₄ treatment, 80 mg Zn/kg Zn-Gly treatment significantly decreased (P < 0.05) the late stage embryo mortality and increased (P < 0.05) liver Zn concentration in the embryo of d9, d19, and 1-day-old chick. The 80 mg Zn/kg Zn-Gly treatment significantly increased (P < 0.05) copper-zinc superoxide dismutase activity in d19 embryo and 1-day-old chick, total superoxide dismutase activity in 1-day-old chick, and copper-zinc superoxide dismutase messenger RNA (mRNA) abundance of d9 embryo and 1-day-old chick than that in ZnSO₄ treatment. The liver metallothionein concentration of the developing embryo and 1-day-old chick and its mRNA abundance of d19 embryo were also significantly increased (P < 0.05) in the 80 mg Zn/kg Zn-Gly treatment in comparison with ZnSO₄ treatment. In conclusion, maternal Zn supplementation decreased embryo mortalities of the late stage and the whole period by increasing liver Zn concentration and antioxidant status in d19 embryo and 1-day-old chick, and 80 mg Zn/kg from Zn-Gly treatment was the optimum choice.     

Progeny from sperm obtained after ectopic grafting of neonatal mouse testes

Ectopic grafting of testicular tissue is a promising new approach that can be used to preserve testicular function. This technique has been used recently to differentiate the neonatal testes of different species, up to the level of complete spermatogenesis. This approach can be applied successfully to generate live progeny using sperm extracted from grafts originating from testes of newborn donors. The sperm are capable of supporting normal development and producing fertile male and female offspring after intracytoplasmic injection into mouse oocytes and embryo transfer into surrogate mothers. The grafted tissue was also capable of significantly normalizing reproductive hormone levels in the castrated recipients. This technique presents new avenues for experimentation. The recipient mouse can be regarded as a living incubator and a culture system of testicular tissue, allowing the experimental manipulation of several aspects of testis development and spermatogenesis. The successful generation of pups indicates that this technique can be used to study the testicular phenotype and to breed mutant or transgenic mouse strains with lethal postnatal phenotypes. The ability to generate sperm from the germ line ex vivo also paves the way for the development of new strategies for preserving fertility in boys undergoing cancer therapy.     

Developmental regulation of ornithine decarboxylase (ODC) in rat testis: comparison of changes in ODC activity with changes in ODC mRNA levels during testicular maturation.

These studies were undertaken to analyze the changes in testicular ornithine decarboxylase (ODC) mRNA levels and ODC activity in rats from birth to maturity. Levels of ODC mRNA were initially low in animals aged 10-17 days. Beginning at 21 days, ODC mRNA levels began to rise, reaching maximal levels by 40 days (p less than 0.01). The size of the 2.2- and 2.6-kb ODC mRNAs did not appear to change with age, as determined by Northern blot analysis. The increase in ODC mRNA that began at 21 days paralleled the increase in testis weight. This increase in ODC mRNA preceded the appearance of rat protamine-1 mRNA, a germ cell-specific mRNA found in round spermatids, which was first detected on Day 40. In contrast, levels of sulfated glycoprotein-2 mRNA, which, in the testis, is found exclusively in Sertoli cells, were highest at Day 17 and thereafter declined gradually with age. Unlike the increase in ODC mRNA levels, ODC activity was highest in 10-day-old animals and thereafter declined steadily with age, reaching minimal levels by 40 days (p less than 0.01). Thus, the increase in testicular ODC mRNA levels was in marked contrast to the decrease in testicular ODC activity. Incubation of cytosolic extract from 40-day-old animals with that from 10- or 17-day-old animals inhibited ODC activity approximately 50%, when compared to cytosols from 10- or 17-day-old animals. Dialysis of cytosol from 40-day-old animals prior to incubation with cytosol from 10-day-old animals relieved this inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)