Profiles of 21-Carbon Steroids in 21-hydroxylase Deficiency.

CONTEXT: Marked elevations of 17-hydroxyprogesterone (17OHP) are characteristic of classic 21-hydroxylase deficiency (21OHD). Testing of 17OHP provides the basis for 21OHD diagnosis, although it suffers from several pitfalls. False-positive or false-negative results and poor discrimination of nonclassic 21OHD from carriers limit the utility of serum 17OHP and necessitate dynamic testing after cosyntropin stimulation when values are indeterminate. OBJECTIVE: The objective was to provide a detailed characterization of 21-carbon (C21) steroids in classic 21OHD, which might identify other candidate steroids that could be employed for the diagnosis of 21OHD. SETTING AND PARTICIPANTS: Patients (11 women, 10 men) with classic 21OHD and 21 sex- and age-matched controls seen in a tertiary referral center were studied. METHODS: C21 steroids in the peripheral sera from all subjects, as well as in media from cultured testicular adrenal rest tumor (TART) cells and normal adrenal (NA) cells, were analyzed using liquid chromatography/tandem mass spectrometry (10 steroids). Additionally, the dynamics of C21 steroid metabolism in TART and NA cells were assessed with radiotracer studies. RESULTS: Five C21 steroids were significantly higher in 21OHD patients: 17OHP (67-fold; P < .01), 21-deoxycortisol (21dF; 35-fold; P < .01), 16α-hydroxyprogesterone (16OHP; 28-fold; P < .01), progesterone (2-fold; P < .01), and 11ß-hydroxyprogesterone (11OHP; not detected in controls; P < .01). The same steroids were the highest in media from TART cells relative to the NA cells: 11OHP, 58- to 65-fold; 21dF, 30- to 41-fold; 17OHP, 9-fold; progesterone, 9- to 12-fold; and 16OHP, 7-fold. CONCLUSION: Measurement of 16OHP and 11OHP along with 17OHP and 21dF by liquid chromatography/tandem mass spectrometry might comprise a biomarker panel to accurately diagnose all forms of 21OHD.

Bone Morphogenetic Protein-4 (BMP4): A Paracrine Regulator of Human Adrenal C19 Steroid Synthesis.

Bone morphogenetic proteins (BMPs) comprise one of the largest subgroups in the TGF-ß ligand superfamily. We have identified a functional BMP system equipped with the ligand (BMP4), receptors (BMP type II receptor, BMP type IA receptor, also called ALK3) and the signaling proteins, namely the mothers against decapentaplegic homologs 1, 4, and 5 in the human adrenal gland and the human adrenocortical cell line H295R. Microarray, quantitative RT-PCR, and immunohistochemistry confirmed that BMP4 expression was highest in the adrenal zona glomerulosa followed by the zona fasciculata and zona reticularis. Treatment of H295R cells with BMP4 caused phosphorylation of the mothers against decapentaplegic and a profound decrease in synthesis of the C19 steroids dehydroepiandrosterone (DHEA), DHEA sulfate, and androstenedione. Administration of BMP4 to cultures of H295R cells also caused a profound decrease in the mRNA and protein levels of 17α-hydroxylase/17,20-lyase (CYP17A1 and P450c17, respectively) but no significant effect on the mRNA levels of cholesterol side-chain cleavage cytochrome P450 (CYP11A1) or type 2 3ß-hydroxysteroid dehydrogenase (HSD3B2). Furthermore, Noggin (a BMP inhibitor) was able to reverse the negative effects of BMP4 with respect to both CYP17A1 transcription and DHEA secretion in the H295R cell line. Collectively the present data suggest that BMP4 is an autocrine/paracrine negative regulator of C19 steroid synthesis in the human adrenal and works by suppressing P450c17.

The historical Coffin-Lowry syndrome family revisited: identification of two novel mutations of RPS6KA3 in three male patients.

Coffin-Lowry syndrome (CLS) is a rare X-linked dominant disorder characterized by intellectual disability, craniofacial abnormalities, short stature, tapering fingers, hypotonia, and skeletal malformations. CLS is caused by mutations in the Ribosomal Protein S6 Kinase, 90 kDa, Polypeptide 3 (RPS6KA3) gene located at Xp22.12, which encodes Ribosomal S6 Kinase 2 (RSK2). Here we analyzed RPS6KA3 in three unrelated CLS patients including one from the historical Coffin-Lowry syndrome family and found two novel mutations. To date, over 140 mutations in RPS6KA3 have been reported. However, the etiology of the very first familial case, which was described in 1971 by Lowry with detailed phenotype and coined the term CLS, has remained unknown. More than 40 years after the report, we succeeded in identifying deposited fibroblast cells from one patient of this historic family and found a novel heterozygous 216 bp in-frame deletion, encompassing exons 15 and 16 of RPS6KA3. Drop episodes in CLS patients were reported to be associated with truncating mutations deleting the C-terminal kinase domain (KD), and only one missense mutation and one single basepair duplication involving the C-terminal KD of RSK2 in the patients with drop episode have been reported thus far. Here we report the first in-frame deletion in C-terminal KD of RPS6KA3 in a CLS patient with drop episodes.

The molecular basis of impaired follicle-stimulating hormone action: evidence from human mutations and mouse models.

The pituitary gonadotropin follicle-stimulating hormone (FSH) interacts with its membrane-bound receptor to produce biologic effects. Traditional functions of FSH include follicular development and estradiol production in females, and the regulation of Sertoli cell action and spermatogenesis in males. Knockout mice for both the ligand (Fshb) and the receptor (Fshr) serve as models for FSH deficiency, while Fshb and Fshr transgenic mice manifest FSH excess. In addition, inactivating mutations of both human orthologs (FSHB and FSHR) have been characterized in a small number of patients, with phenotypic effects of the ligand disruption being more profound than those of its receptor. Activating human FSHR mutants have also been described in both sexes, leading to a phenotype of normal testis function (male) or spontaneous ovarian hyperstimulation syndrome (females). As determined from human and mouse models, FSH is essential for normal puberty and fertility in females, particularly for ovarian follicular development beyond the antral stage. In males, FSH is necessary for normal spermatogenesis, but there are differences in human and mouse models. The FSHB mutations in humans result in azoospermia; while FSHR mutations in humans and knockouts of both the ligand and the receptor in mice affect testicular function but do not result in absolute infertility. Available evidence also indicates that FSH may also be necessary for normal androgen synthesis in males and females.

Gene expression of 11ß-HSD and glucocorticoid receptor in the bovine (Bos taurus) follicle during follicular maturation and atresia: the role of follicular stimulating hormone.

Glucocorticoids modulate ovarian function in cattle. However, their regulatory mechanisms have not been fully elucidated. In the present study, we examined gene expression of two glucocorticoid-metabolizing enzymes, a bidirectional 11ß-HSD type 1 (11HSD1) and a dehydrogenase 11ß-HSD type 2 (11HSD2), and glucocorticoid receptor (GR) in bovine follicles during follicular maturation and atresia. Granulosa cells (GCs) and theca interna layers (TIs) were harvested from follicles classified as small growing, dominant, preovulatory, early atretic and late atretic follicles. The expression levels of 11HSD1, 11HSD2 and GR mRNA were quantified by real-time PCR. In the healthy follicles, expression of 11HSD1 mRNA increased as follicles matured, both in GCs and TIs. A significant negative correlation was found between the concentration of cortisol in follicular fluid and the level of 11HSD1 mRNA in GCs. The expression of 11HSD2 and GR was either very low or largely unchanged during follicular maturation. In the atretic follicles, a drastic increase in the expression of 11HSD2 was observed both in GCs and TIs. To assess the effect of FSH on the expression of 11HSDs and GR, GCs were cultured with FSH (0-100 ng/ml) for up to 6 days. FSH increased 11HSD1 mRNA in a dose-dependent manner, but not 11HSD2, nor GR. Taken together, these results suggest that developmentally-regulated 11HSD1 plays a pivotal role in modulating the local glucocorticoid environment in maturing bovine follicles.

Classification of bovine follicles based on the concentrations of steroids, glucose and lactate in follicular fluid and the status of accompanying follicles.

A simple and clear means to identify the physiological status of follicles is essential for study of follicular biology. In the present study, we verified a novel classification procedure based on analysis of the follicular population and glucose concentration in follicular fluid (FF) as an alternative method to classify bovine follicles. Paired ovaries were collected from heifers, and the number of follicles and stage of the CL were recorded. Follicles were initially divided into the following 3 groups according to diameter and the ratio of E2 and P4 (E/P): E2 active (E-A: E/P>or=1), E2 inactive (E-I: E/P<1, >or=8.5 mm) and small follicles (E/P<1, <8.5 mm). E-A follicles were easily identified as E2-rich dominant follicles and were further classified according to diameter and stage of the CL as early dominant (EDF: <8.5 mm), dominant (DF: >or=8.5 mm, stages I-III) or preovulatory follicles (POF: >or=8.5 mm, stage IV). E-I follicles were classified as follows based on the status of the accompanying follicles: early atretic (EAF: without an E-A follicle), mid-atretic (MAF: with an EDF or DF) and late atretic follicles (LAF: with an EAF or POF). The follicular P4 concentrations of the MAF and LAF were significantly higher compared with that of the EAF, while follicular glucose concentration of the LAF was lower compared with those of EAF and MAF, indicating that this classification can be used to distinguish early atretic follicles from more advanced atretic follicles. Small follicles were classified as growing (GF: without E-A follicles) and suppressed small follicles (SSF: with E-A follicles). The SSF was easily identifiable by this procedure, but some GF populations likely contained SSF. To identify true GF, the ratio of E2 in the GF and accompanying EAF may be used. In conclusion, analysis of the follicular population in conjunction with biochemical indices such as steroid and glucose concentrations in FF provides a simple and accurate means of classifying bovine follicles.

Hormonal regulation of expression of growth differentiation factor-9 receptor type I and II genes in the bovine ovarian follicle.

Growth differentiation factor-9 (GDF-9) and bone morphogenetic proteins (BMPs) are crucial factors in follicular growth and development. GDF-9 and BMPs initiate signaling by assembling type I (ALK-3, ALK-5 and ALK-6) and type II (BMPRII) receptors. However, the mechanism regulating the expression of these receptors in the process of bovine follicle development is still unknown. The aim of the present study was to clarify the involvement of receptor systems for GDF-9 and BMPs in follicular selection by examining the effects of FSH and estradiol-17beta (E2) on the regulation of BMPRII, ALK-3, ALK-5 and ALK-6 mRNA expression in bovine granulosa cells (GCs). To observe mRNA expression during follicular development, follicles were obtained from heifers and classified into two groups: pre-selection follicles (PRFs) (an average of 7.7 mm follicles with low E2) and post-selection follicles (POFs) (an average of 15 mm follicles with high E2). Theca layer cells (TCs) and GCs were harvested from aspirated follicles. For in vitro studies, GCs were obtained from bovine follicles of 4-7 mm diameter and cultured in Dulbecco's modified Eagle's/F12 (DMEM/F-12) medium with 10% fetal calf serum for 24 h. The medium was then replaced with serum-free DMEM/F-12 supplemented with different doses of E2 (1, 10, 100 ng/ml) or FSH (1, 5, 10 ng/ml) or combinations of 1 ng/ml of E2 with different FSH doses. Total RNA was extracted and the mRNA expression of BMPRII, ALK-3, ALK-5 and ALK-6 was estimated by the quantitative real-time PCR method using a LightCycler. BMPRII and ALK-5 expression was significantly higher in the GCs of POFs than in those of PRFs, whereas ALK-3 expression was significantly lower in the GCs of POFs than in those of PRFs. There was no difference in ALK-6 expression in GCs between PRFs and POFs. The expression of BMPRII, ALK-5, ALK-3 and ALK-6 genes in the TCs was not significantly different between PRFs and POFs. Treatment of GCs with E2 alone increased BMPRII mRNA expression at a concentration of 100 ng/ml and ALK-5 mRNA expression at 10 ng/ml. BMPRII and ALK-5 mRNA levels were up-regulated by the combination of E2 (1 ng/ml) and FSH (5 ng/ml). On the other hand, FSH alone down-regulated the expression of BMPRII and ALK-5 in GCs. The results of the present study provide the first evidence that FSH and E2 regulate the expression of BMPRII and ALK-5 genes in bovine GCs. Thus, our data suggest that the GDF-9/BMPRII/ALK-5 system may be critically involved in the process of selection of bovine follicles.

Hormonal regulation and differential expression of neuropilin (NRP)-1 and NRP-2 genes in bovine granulosa cells.

Although much is known about the biology of vascular endothelial growth factor (VEGF) and its receptors, little is known about the role of the VEGF receptors neuropilin (NRP)-1 and NRP-2 in the process of bovine follicle development. The aim of the present study was to examine the hormonal regulation of NRP-1 and NRP-2 mRNAs by real-time PCR in follicles from the bovine ovary and in cultured granulosa cells. The NRP-1 gene was expressed in the granulosa and theca cells in the post-selection (POF) and pre-selection (PRF) follicles in the bovine ovary. In contrast, the NRP-2 gene was expressed only in the theca cells in the POF and the PRF. The level of NRP-1 mRNA was significantly increased by treatment of the cultured granulosa cells with 10 ng/ml estradiol (E2). In contrast, the addition of progesterone (P4) to the culture medium decreased the expression of the NRP-1 gene. The level of NRP-1 mRNA was increased by 10 ng/ml E2 with or without 1 ng/ml P4, but the level of NRP-1 mRNA was decreased if the P4 level was increased to 10 ng/ml, even when 1 ng/ml E2 was also added. Follicle-stimulating hormone did not stimulate the expression of the NRP-1 gene. These results are the first data showing that NRP-1, but not NRP-2, is expressed in the granulosa cells of bovine follicles and that NRP-1 gene expression is regulated by sex steroids. Our findings suggest the involvement of NRP-1 in follicle development in the cow.

Involvement of the bone morphogenetic protein/receptor system during follicle development in the bovine ovary: Hormonal regulation of the expression of bone morphogenetic protein 7 (BMP-7) and its receptors (ActRII and ALK-2).

Bone morphogenetic proteins (BMPs) are crucial factors in follicular growth and development. Among the BMP ligands, BMP-7 which use ActRII as their type II receptor, strongly bind to ALK-2 as their type I receptor. However, whether their receptors are expressed and the regulatory mechanisms controlling their expression during the process of bovine follicle development are still unknown. The aim of the present study was to clarify the involvement of the receptor system for BMP-7 in follicular selection by examining the effects of follicle-stimulating hormone (FSH) and estradiol (E2) on the regulation of ActRII and ALK-2 mRNA expression in bovine granulosa cells (GCs). To observe mRNA expression, follicles were obtained from heifers and GCs were classified into two groups: pre-selection follicles (PRF; follicles with an average diameter of 7 mm and low E2) and post-selection follicles (POF; follicles with an average diameter of 15 mm and high E2). The theca cell (TC) layer and GCs were harvested from aspirated follicles. For in vitro studies, GCs were obtained from bovine follicles of 4-7 mm diameter and cultured in Dulbecco's modified Eagle's/F12 (DMEM/F-12) medium with 10% fetal calf serum for 24h. The medium was then replaced with serum-free DMEM/F-12 supplemented with different doses of E2 (1, 10,100 ng/ml), FSH (1, 5, 10 ng/ml) or combinations of 1 ng/ml of E2 with different FSH doses (1, 5, 10 ng/ml). Total RNA was extracted from GCs and the mRNA expression of ActRII and ALK-2 was estimated by the quantitative PCR method using LightCycler. The expression of BMP-7 mRNA in TCs did not differ between the PRF and POF. ActRII and ALK-2 expression was detected in GCs from bovine antral follicles and was higher in the GCs of POF than in those of PRF, while the expression of the ActRII and ALK-2 genes in the TCs was not different between PRF and POF. Treatment of GCs with E2 (10 ng/ml) alone increased the expression of both ActRII and ALK-2 mRNAs, whereas FSH alone had no effect. However, ActRII and ALK-2 mRNA levels were up-regulated by the combination of E2 (1 ng/ml) and FSH (5 ng/ml). The results of the present study provide the first evidence that FSH and E2 regulate the expression of the ActRII and ALK-2 genes in bovine GCs. Thus, our data suggest that the BMP7/ActRII/ALK-2 system may be critically involved in the process of selection of bovine follicles.

Relationship between ovarian weight and follicular population in heifers.

The size of the ovary varies substantially among cattle. This variation may influence the potential of the ovary to produce follicles. In the present study, we examined whether a relationship exists between the weight of the ovary and the number of antral follicles >or=1 mm. Paired ovaries were obtained from Holstein x Japanese Black F1 heifers. Follicles were classified into three size categories (small: 1.0-<5.0 mm, medium: 5.0-<8.5 mm and large: >or=8.5 mm), and the number of follicles in each category was recorded. Large variations in the weight of ovaries and the number of follicles were observed among animals. Significant positive correlations (r>or=0.4, P<0.001) were found between the weight of intact ovaries and the number of follicles in all three categories for the ovary contralateral to CL (OCC) and in the small follicles for the ovary ipsilateral to CL (OIC). Significant positive correlations (r>0.4, P<0.0001) were also observed between the weight of ovaries devoid of CL and follicles and the number of small and medium follicles in both OIC and OCC, indicating that the correlation is not due to the increase in ovarian weight associated with the increase in follicular number. Paired ovaries contained a similar number of small and medium follicles, and significant positive correlations were observed between them (r>0.6, P<0.0001). There were significant positive correlations between the weight of OCC and the number of small and medium follicles in paired ovaries (r>0.4, P<0.0001). These results suggest that 1) the weight of an ovary reflects the potential of the ovary to produce antral follicles, and 2) a rough estimation of follicular population might be possible by using the weight of the ovary contralateral to CL in heifers.