Immunodetection and quantification of enzymatic markers in theca cells: the early process of ovarian steroidogenesis†

The association between theca cells (TCs) and granulosa cells is pivotal to steroid biosynthesis in the ovary. During the late secondary follicle stage, TCs form a layer around granulosa cells, after which their steroidogenic function falls under the control of luteinizing hormone (LH) that activates the cAMP signaling pathway via a G protein-coupled receptor. In addition to perilipin-2, a marker for lipid droplets containing esters as substrates for TCs to produce steroidogenic hormones, other essential proteins, like steroidogenic acute regulatory protein (StAR), cytochrome P450 11A1, cytochrome P450c17, 3 beta-hydroxysteroid dehydrogenase/delta 5 —> 4-isomerase type 1, and 3 beta-hydroxysteroid dehydrogenase/delta 5 —> 4-isomerase type 2, play a role in the cascade after luteinizing hormone–choriogonadotropic hormone receptor (LH/CG-R) occupation by LH. The aim of the present study was to assess expression levels and corresponding amounts of LH/CG-R, perilipin-2, and enzymes involved in the steroidogenic pathway of TCs based on follicle stage. Immunohistochemical analysis of each of these proteins was therefore performed on ovarian samples from nine adult women, most (n = 8) with BRCA1 and/or BRCA2 mutations undergoing prophylactic bilateral oophorectomy. Pictures were taken of the theca layer of secondary, small (<3000 μm), and large (>3000 μm) antral follicles and corpora lutea at 100× magnification. ImageJ software was used to analyze the surface area and expression intensity of each protein at each stage, known as the staining index. Overall, our data showed that LH/CG-R, perilipin-2, and StAR expression increased in the course of folliculogenesis and luteinization. Similarly, cytochrome P450 11A1, cytochrome P450c17, 3 beta-hydroxysteroid dehydrogenase/delta 5 —> 4-isomerase type 1, and 3 beta-hydroxysteroid dehydrogenase/delta 5 —> 4-isomerase type 2 expression were substantially elevated in TCs during folliculogenesis, evidenced by their coordinated action in terms of area covered and expression intensity. This study, conducted for the first time on human ovarian tissue, contributes to localizing and quantifying expression of key steroidogenic proteins at both intracellular and tissue levels. These findings may shed new light on pathological conditions involving the human ovary, such as androgen-secreting tumors of the ovary and other disorders associated with ovarian TCs in patients with polycystic ovary syndrome.

Adrenal glands of Spix's yellow-toothed cavy (Galea spixii, Wagler, 1831): morphological and morphometric aspects

Considering the physiological importance and need of greater morphophysiological knowledge of adrenal glands, the aims of present study were compare the morphometric data between left and right adrenal of male and female; perform a histological, scanning and transmission electron microscopy study showing tissue constitution of glands; finally, in order to define the presence and correct site of the cytochrome P450c17 expression in adrenal glands, immunohistochemical study of this enzyme was performed in 18 adrenal glands (right n=9 and left n=9) of nine adult Galea spixii (four males and five females). Right adrenal was more cranially positioned than left adrenal; dimensions (weight, length and width) of right adrenal was larger than left adrenal; no differences between male and female body and adrenal measurements were found; the morphology of cells and different amounts of lipid droplets may be related to the different demands of steroid hormones production, related to each zone of the adrenal cortex; and, the cytochrome P450c17 immunolocalization in fasciculate and reticular zone may be related with synthesis of 17-hydroxy-pregnenolone, 17-hydroxy-progesterone, dehydroepiandrosterone or androstenedione.

68 STEROIDOGENIC CONTROL OF INTRAUTERINE SEXUAL DIFFERENTIATION IN SPIX’S YELLOW-TOOTHED CAVY, GALEA SPIXII

The Spix’s yellow toothed (SYT) cavy is a species of rodent that lives in Caatinga vegetation of northeast Brazil. The SYT is utilised as a protein source by inhabitants of that region and has zootechnic potential. SYT cavies have been bred in captivity for studies related to reproductive biology. The presence of a penile clitoris trespassed by the urethra was described in the adult female. The aim of this research is describe sexual differentiation events in the male and female conceptus and relate molecular mechanisms of androgen and oestrogen metabolism of the placenta with the differentiation of external genitalia. Conceptuses after 15, 22, 30, 40, >50 (full term) days of gestation (DG; n = 9 males and 9 females at each gestational period) from 30 pregnant females, which were paired with 10 males, were used. After detection of copulation, the above described DG were awaited prior to the day of euthanasia. We performed anatomical description and light and scanning electron microscopy on the genital organs in the conceptus; concentration of testosterone (T) and oestradiol (E) by radioimmunoassay in the pregnant females; and described the immunoreactivity (by immunoblotting and immunohistochemistry) of enzymes: cytochrome P450 aromatase, cytochrome P450c17, 17-β-HSD, and 3-β-HSD in placentas of male and female conceptus. The genital organs emerge from a pair of undifferentiated gonads and a genital tubercle at 15 DG. These gonads differentiate into a pair of testicles or a pair of ovaries at 22 DG. The other internal genital organs arise from the urogenital sinus and Müllerian ducts in females or Wolffian ducts in males after 22 DG. The undifferentiated genital tubercle forms the penis in males with the urethra channeling at 30 DG. In females, the urethra channeling is complete after 40 DG with the closure of the urethral groove, and at >50 DG the clitoris has characteristics of a pseudo-penis. Those that are pregnant undergo an increase of E (from 5.456 ± 1.599 pg mL–1 at 22 DG to 107.186 ± 15.966 pg mL–1 at >50 DG) and T (from 122.444 ± 15.799 pg mL–1 at 22 DG to 718.555 ± 67.407 pg mL–1 at >50 DG) concentrations during the gestational period. The concentration of T was a minimum 5-fold higher than the E concentration at the moment of external sexual differentiation. The placentas from the male and female conceptus are immunoreactive for the enzyme 3-β-HSD producing progesterone throughout pregnancy, but these placentas are not immunoreactive for the oestrogen-producing enzyme cytochrome P450 aromatase throughout pregnancy. Conversely, the cytochrome P450c17 and 17-β-HSD enzymes are immunoreactive in the placentas of the male and female conceptus throughout pregnancy, producing androgen hormones. The absence of enzymes that produce oestrogen hormones in the placenta of SYT cavies indicate that testosterone from pregnant reaches the fetal circulation, and associated with the presence of enzymes that produce androgens, may act to increase androgen hormone concentration in the pregnant, which may act in the virilization process of the female external genitalia.