Structural proteins in sexual differentiation of embryonic gonads

Sexual differentiation of embryonic gonads was studied by immunocytochemical analysis of cytoskeleton, basement membrane and extracellular matrix. The epithelial cells of the prospective gonadal region in both sexes contained vimentin and desmin intermediate filament proteins but not cytokeratin. Basement membrane components laminin, collagen types IV and V, heparan sulfate proteoglycan, and fibronectin were seen in an unorganized form in the extracellular space. The development of the gonads started by proliferation of the pregonadal epithelial cells, which formed separate clusters and loose mesenchyme. In the male gonad the clusters joined together into elongated cords, outlined by basement membrane components. The cord cells became polarized epithelial cells, and cytokeratin appeared with the disappearance of desmin in their cytoplasm. Desmin and vimentin remained in the interstitial cells. In the female gonad the clusters were smaller, and the cords were irregular in shape and size. Desmin disappeared from the cord cells and cytokeratin appeared, but more slowly and less well polarized than in the testis. The present results show that after common early development, the sexual differences in gonads emerge as different organization of the internal epithelial tissue with different timing of changes in intra- and extracellular components.     

Ultrasonographic observations of the maturation of basic movements in guinea pig fetuses

Ultrasonography has not previously been used for studying fetal movements in precocial rodents. The objective of this study was to ultrasonographically determine the sequence of the appearance of basic movements in a guinea pig fetus. The research included eight guinea pig females carrying one fetus each. Fetal movements were observed for 10 minutes each day, from the 25th to 38th day of gestation. The time and sequence of the appearance of movements was observed as follows: whole body flexion (mean 27.6 SD ± 1.68), whole body extension (mean 28.1 SD ± 1.12), head flexion (mean 28.1 SD ± 1.80), head extension (mean 30.5 SD ± 2.67) forelimbs flexion (mean 30.5 SD ± 2.32), forelimbs extension (mean 30.7 SD ± 1.84), trunk rotation (mean 31.9 SD ± 2.23), forelimbs alternating flexion and extension (mean 32.1 SD ± 2.1), hind limbs extension (mean 32.2 SD ± 3.2), hind limbs flexion (mean 32.4 SD ± 3.16), and hind limbs alternating flexion and extension (mean 33.5 SD ± 2.39). The identical sequences of basic movement appearances in guinea pigs, sheep, and rats suggest that the rostrocaudal gradient of basic movement appearance could be a general developmental pattern in mammalian species.     

Cerebral energy metabolism in guinea pig fetuses during development.

During development fetal arterial oxygen tension falls, whereas cerebral oxygen consumption rises due to an increase in cerebral metabolism. To compensate for this increase in oxygen consumption, blood flow and therefore oxygen delivery to the cerebrum rises. To determine whether during development oxygen delivery to the cerebrum meets cerebral oxygen consumption, we measured the concentrations of high-energy phosphates and glycolytic intermediates in the cerebral cortex of fetal guinea pigs at different gestational ages. During development there was no change in the concentrations of adenosine triphosphate, creatine phosphate, adenosine monophosphate, and lactate. However, cerebral concentrations of adenosine diphosphate increased and those of glucose decreased. Our results suggest that the increase in fetal cerebral oxygen delivery during development meets cerebral oxygen consumption with increasing gestational age. We speculate that the measured rise in the concentrations of adenosine diphosphate may accelerate glycolysis during development and therefore may cause a rise in both cerebral blood flow to maintain oxygen delivery.     

Lectin-binding carbohydrates in sexual differentiation of rat male and female gonads

Summary Development and sexual differentiation of the mammalian gonad involve changes in the type and distribution of different proteins and glycoproteins in and around the epithelial gonadal cords, the future seminiferous tubules in the testis, and follicles in the ovary. To study changes in cellular carbohydrate-containing compounds in the sex-specific morphogenesis of rat gonads, sections from embryonic, fetal and early postnatal gonads were labelled with seven different fluorescein isothiocyanate (FITC)-conjugated plant lectins of various carbohydrate-binding specificities. Double labelling of laminin with tetramethylrhodamine isothiocyanate (TRITC)-conjugated antibodies was used to outline the epithelial tissues. From the results we conclude that the abundance and similar distribution of carbohydrates in the early gonads of both sexes supports their sexually indifferent nature. Furthermore, the basement membranes of the differentiating gonadal cords in both sexes have common features, which differ, however, from those of the other developing urogenital organs. Changes in carbohydrate composition appear with the sexual differentiation of the gonads; the similarity of the changes in lectin binding to the gonadal cords of embryonic and fetal male, and to postnatal female, suggests similar mechanisms of cell-cell interactions in both sexes although activated at different developmental stages. These carbohydrate specificites at the tissue level should be taken into account together with cell-type specific changes, e.g. in the formation of the zona pellucida, when the phenomenon of polymorphic expression of different compounds is integrated into theories of epithelial differentiation.     

Sex determination and sexual differentiation in the avian model

The sex of birds is determined by the inheritance of sex chromosomes (ZZ male and ZW female). Genes carried on one or both of these sex chromosomes control sexual differentiation during embryonic life, producing testes in males (ZZ) and ovaries in females (ZW). This minireview summarizes our current understanding of avian sex determination and gonadal development. Most recently, it has been shown that sex is cell autonomous in birds. Evidence from gynandromorphic chickens (male on one side, female on the other) points to the likelihood that sex is determined directly in each cell of the body, independently of, or in addition to, hormonal signalling. Hence, sex-determining genes may operate not only in the gonads, to produce testes or ovaries, but also throughout cells of the body. In the chicken, as in other birds, the gonads develop into ovaries or testes during embryonic life, a process that must be triggered by sex-determining genes. This process involves the Z-linked DMRT1 gene. If DMRT1 gene activity is experimentally reduced, the gonads of male embryos (ZZ) are feminized, with ovarian-type structure, downregulation of male markers and activation of female markers. DMRT1 is currently the best candidate gene thought to regulate gonadal sex differentiation. However, if sex is cell autonomous, DMRT1 cannot be the master regulator, as its expression is confined to the urogenital system. Female development in the avian model appears to be shared with mammals; both the FOXL2 and RSPO1/WNT4 pathways are implicated in ovarian differentiation.     

Lectin-binding carbohydrates in sexual differentiation of rat male and female gonads.

Development and sexual differentiation of the mammalian gonad involve changes in the type and distribution of different proteins and glycoproteins in and around the epithelial gonadal cords, the future seminiferous tubules in the testis, and follicles in the ovary. To study changes in cellular carbohydrate-containing compounds in the sex-specific morphogenesis of rat gonads, sections from embryonic, fetal and early postnatal gonads were labelled with seven different fluorescein isothiocyanate (FITC)-conjugated plant lectins of various carbohydrate-binding specificities. Double labelling of laminin with tetramethylrhodamine isothiocyanate (TRITC)-conjugated antibodies was used to outline the epithelial tissues. From the results we conclude that the abundance and similar distribution of carbohydrates in the early gonads of both sexes supports their sexually indifferent nature. Furthermore, the basement membranes of the differentiating gonadal cords in both sexes have common features, which differ, however, from those of the other developing urogenital organs. Changes in carbohydrate composition appear with the sexual differentiation of the gonads; the similarity of the changes in lectin binding to the gonadal cords of embryonic and fetal male, and to postnatal female, suggests similar mechanisms of cell-cell interactions in both sexes although activated at different developmental stages. These carbohydrate specificities at the tissue level should be taken into account together with cell-type specific changes, e.g. in the formation of the zona pellucida, when the phenomenon of polymorphic expression of different compounds is integrated into theories of epithelial differentiation.     

Sex differentiation and pubertal development of gonads in the viviparous mosquitofish, Gambusia affinis

The ontogenetic development of gonads from embryo to adult was observed histologically in the viviparous teleost, Gambusia affinis. Primordial germ cells (PGCs) appeared in the subendodermal space of the embryo 14 days before birth, and then transferred to the dorsal mesentery to form paired genital ridges 12 days before birth. The PGCs proliferated in the genital ridge, forming gonadal primordia 10 days before birth. All gonadal primordia differentiated to the ovary containing oocytes 2 days before birth, but then redifferentiated to the ovary and testis just after birth. This indicates that the mosquitofish is a juvenile hermaphroditic species. The characteristics of gonadal sex differentiation just after birth were enlargement of the oocytes in females, and invasion of somatic cells from the hilar region to an inner portion of the gonad in males. The paired ovary fused at the basal area 5 days after birth, then on the ventral and dorsal portions, developing into a single ovary 10 days after birth. During this time a single ovarian cavity was formed on the dorsal portion of the ovary. The paired testes fused only at the basal area and became a single testis having two main lobes 10 days after birth. The oocytes gradually developed and began vitellogenesis 100 days after birth, but did not reach maturation until 110 days after birth. Spermatogenic cells formed cysts at 20 days, began meiosis at 70 days, and matured to form sperm balls 90 days after birth. The male fish sexually matured earlier than the female.     

Steroid hormone content of the gonads of the tammar wallaby during sexual differentiation.

The gonads of the tammar wallaby, Macropus eugenii, are sexually indifferent at birth (Day 0) despite the fact that phenotypic sexual differentiation has already commenced as evidenced by the presence of a scrotum in males and mammary anlagen in females. The seminiferous cords of the testis first become clearly recognizable on Day 2 of pouch life, and ovarian differentiation is recognizable by Day 10. To monitor the endocrine development of the gonads during sexual differentiation of the urogenital tract, we measured the steroid hormone content in 92 pools of gonads from male and female tammar pouch young from the day of birth to 206 days of pouch life. Progesterone, estradiol, and dihydrotestosterone concentrations were low (less than 0.05 ng/mg protein) in both ovaries and testes at all stages examined, and testosterone concentrations were uniformly low in ovaries. Testosterone concentrations in testes were low on Days 0-4, averaging about 0.2 ng/mg protein; they rose by Days 5-10 to an average of 0.9 ng/mg protein, remained elevated until about Day 40, and thereafter fell to values similar to those in the ovaries. The phallus and urogenital sinus were able to convert testosterone to dihydrotestosterone from the earliest stages examined (Days 10 and 11). Thus in the tammar wallaby, as in eutherian mammals, testosterone is the androgen secreted by the developing testis, and dihydrotestosterone is formed in certain androgen target tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of exogenous steroids on androgen receptors in fetal guinea pig brain

We treated pregnant guinea pigs on Day 50 of gestation with 10 mg testosterone propionate (TP), obtaining fetuses 2, 4, 8, or 18 h later as well as after 5 days of treatment. In a second group of pregnant guinea pigs, dihydrotestosterone propionate (DHTP), estradiol benzoate (E2B), progesterone (P), or cortisol was given 2 h before obtaining fetuses. Although TP treatment elevated fetal serum T (p less than 0.05), brain cytosolic androgen receptor (ARc) content was unchanged in fetuses of either sex. In female fetuses, nuclear androgen receptors (ARn) increased 10-fold in medial-basal hypothalamus (MBH) and preoptic area (POA) at 2 and 4 h (respectively) after treatment, while fetal male ARn content was unchanged. Maternal injection of other steroids (E2B, P, or cortisol, but not DHTP) significantly increased these hormones in the fetus 2 h later (p less than 0.05). Only androgens affected fetal androgen receptor (AR) content. While TP increased ARn in female MBH, DHTP decreased ARc in fetal anterior pituitary of both sexes. In this latter case, a metabolite of DHT may mediate the effects. We conclude that T crosses the guinea pig placenta and activates ARn in POA and MBH of female fetuses; male ARn appear to be maximally occupied by endogenous T. Steroids of other classes do not induce AR responses in fetal guinea pig brain. These AR changes may represent an initial cellular mechanism in brain sexual differentiation.     

Changes in serum and ovarian steroids during reproductive development in the female guinea pig

This study was designed to measure ovarian hormones prior to and during the first estrous cycle in guinea pigs. Blood was obtained from 12 animals throughout the first estrous cycle. Ovaries and peripheral serum were obtained from 25 additional animals at various stages of development prior to and after first ovulation. Estradiol, progesterone, androstenedione, and testosterone were measured in all sera and half of the ovaries. The remaining ovaries were fixed for histology. Serum estradiol was nondetectable until a few days before first ovulation, but was present in the ovary throughout development. Serum progesterone was nondetectable until the day of ovulation, but the luteal phase pattern was similar to that observed in adults. Serum androgens were detectable throughout development, with androstenedione higher than testosterone. The immature ovary contained more testosterone than androstenedione, but this pattern was reversed after ovulation. These results indicate that the immature ovary in the guinea pig contains minimal amounts of estradiol and progesterone, the first estrous cycle is similar to that in adults, and that the pattern of ovarian androgen content changes during the peripubertal period.     

Involvement of oestrogens in sexual differentiation of gonads as a function of temperature in turtles

Abstract. To investigate the possible involvement of oestrogens in the phenomenon of temperature sex-reversal in the turtle Emys orbicularis , the effects of oestrone, oestradiol and an antioestrogen. tamoxifen, on sexual differentiation of gonads were examined at a male-producing temperature of 25° C and at a female-producing temperature of 30° C. When oestrone or oestradiol were injected into eggs incubated at 25° C just before or at the beginning of the thermosensitive period, the gonads differentiated into ovaries instead of testes. Conversely, when tamoxifen was injected, at the same stages, into eggs incubated at 30° C, epithelial cords or tubes, similar to potential seminiferous cords, differentiated in the interior part of the gonads. However, an ovarian-like cortex persisted at their surface. At 25° C, treatment with tamoxifen or with both tamoxifen and oestradiol also resulted in differentiation of ovotestes. These experiments show that tamoxifen binding to oestrogen receptors prevented the inhibitory action of oestrogens on testicular cord development. Maintenance (at 30° C) or development (at 25° C) of ovarian cortex in the presence of tamoxifen can be expected from an agonistic action of this drug, as already described. Preliminary data on steroid content in the gonads indicate that, during the early stages of the thermosensitive period, the level of estrogens is higher at 30° C than at 25° C. It is proposed that in species displaying temperature sensitivity for the sexual differentiation of gonads, temperature acts on the processes regulating the synthesis or the activity of cyto-chrome-P450 aromatase.     

Sex determining genes and sexual differentiation in a marsupial.

The role of genes in the differentiation of the testis and ovary has been extensively studied in the human and the mouse. Despite over a decade of investigations, the precise roles of genes and their interactions in the pathway of sex determination are still unclear. We have chosen to take a comparative look at sex determination and differentiation to gain insights into the evolution and the conserved functions of these genes. To achieve this, we have examined a wide variety of eutherian sex determining genes in a marsupial, the tammar wallaby, to determine which genes have a conserved and fundamental mammalian sex determining role. These investigations have provided many unique insights. Here, we review the recent molecular and endocrine investigations into sexual development in marsupials, and highlight how these studies have shed light on the roles of genes and hormones in mammalian sex determination and differentiation.     

P450 aromatase expression in the temperature-sensitive sexual differentiation of salamander (Hynobius retardatus) gonads

Sex differentiation of gonads in amphibians is believed to be controlled genetically, but altered epigenetically or environmentally. When larvae of the salamander Hynobius retardatus were reared at defined temperatures from hatching to metamorphic stages, a high temperature (28 degrees C) induced exclusively female gonads (ovaries), whereas intermediate (20 and 23 degrees C) or lower (16 degrees C) temperatures produced a 1:1 sex ratio of the morphological gonads. The thermosensitive period was determined to be restricted from 15 to 30 days after hatching, just before or when sexual differentiation occurred. Hynobius P450 aromatase (P450arom) cDNA was isolated from adult gonads and the partial nucleotide or deduced amino acid sequences were determined, showing a high level of identity with various vertebrate species. The P450arom gene was expressed predominantly in the adult ovary and brain, weakly in testis, but not in other somatic organs. A typical sexual dimorphism in P450arom expression was detected in normally developing larvae by a quantitative competitive RT-PCR; strong expression in the female gonads but very weak in male gonads. The dimorphism was detected much earlier than the morphological sexual differentiation of the gonads. When larvae were reared at the female-producing temperature (28 degrees C), strong expression was detected in all the temperature-treated larvae, suggesting that P450arom was up-regulated, even in genetic males. Our results confirm the importance of the P450arom regulation in the sexual differentiation of gonads and demonstrate that an up-regulation of P450arom is involved in the process of temperature-sensitive sex reversal in this species.     

Unconjugated and sulfoconjugated steroids in plasma and zones of the adrenal cortex of the guinea pig

The following steroids were measured in their unconjugated and sulfoconjugated forms in plasma and in the outer and inner zones of the adrenal cortex of the guinea pig: pregnenolone, 17-hydroxypregnenolone, 21-hydroxypregnenolone, dehydroepiandrosterone and deoxycorticosterone. In plasma, pregnenolone and 21-hydroxypregnenolone were the predominant unconjugated steroids with concentrations 10-30 times higher than the other three steroids. Among the sulfoconjugated steroids, pregnenolone sulfate had a concentration 25-50 times higher than the other sulfoconjugates. For each steroid except 21-hydroxypregnenolone the sulfoconjugated form was present in a concentration 2-7 times higher than the unconjugated form. In the adrenal cortex, the content of 21-hydroxypregnenolone was significantly higher in the outer zone than in the inner zone and was present in amounts 3-100 times greater than the other unconjugated steroids in the outer zone. On the other hand, the content of pregnenolone was significantly greater in the inner zone than the outer zone, and was present in amounts 3-80 times greater than the other unconjugated steroids in the inner zone. With the exception of 21-hydroxypregnenolone and deoxycorticosterone, the steroid sulfoconjugates were significantly higher in the inner cortical zone. As in plasma, pregnenolone sulfate was the most abundant sulfoconjugated steroid. This report also describes preliminary studies concerning sulfurylated hydroxyl groups in different positions of 21-hydroxypregnenolone. The sulfoconjugate was prepared by using partially purified steroid sulfotransferase from the guinea pig adrenal. The results obtained indicated that of the total 21-hydroxypregnenolone conjugate formed, approximately 40% was the 21-sulfate and 20% the 3-sulfate, whereas 40% was non-hydrolyzable with the techniques used and was not further characterized.