Glucose is the main energy substrate in the bovine ovary, and a sufficient supply of it is necessary to sustain the ovarian activity. Glucose cannot permeate the plasma membrane, and its uptake is mediated by a number of glucose transporters (GLUT). In the present study, we investigated the gene expression of GLUT1, 3 and 4 in the bovine follicle and corpus luteum (CL). Ovaries were obtained from Holstein Japanese Black F1 heifers. Granulosa cells and theca interna layers were harvested from follicles classified into five categories by their physiologic status: follicular size (d8·5 mm: dominant; <8·5 mm: subordinate), ratio of estradiol (E 2 ) to progesterone in follicular fluid (d1: E 2 active;<1: E 2 inactive), and stage of estrous cycle (luteal phase, follicular phase). CL were also classified by the stage of estrous cycle. Expression levels of GLUT1, 3 and 4 mRNA were quantified by a real-time PCR. The mRNA for GLUT1 and 3 were detected in the bovine follicle and CL at comparable levels to those in classic GLUT-expressing organs such as brain and heart. Much lower but appreciable levels of GLUT4 were also detected in these tissues. The gene expression of these GLUT showed tissue-and stage-specific patterns. Despite considerable differences in physiologic conditions, similar levels of GLUT1, 3 and 4 mRNA were expressed in subordinate follicles as well as dominant E 2 -active follicles in both luteal and follicular phases, whereas a notable increase in the gene expression of these GLUT was observed in dominant E 2 -inactive follicles undergoing the atretic process. In these follicles, highly significant negative correlations were observed between the concentrations of glucose in follicular fluid and the levels of GLUT1 and 3 mRNA in granulosa cells, implying that the local glucose environment affects glucose uptake of follicles. These results indicate that GLUT1 and 3 act as major transporters of glucose while GLUT4 may play a supporting role in the bovine follicle and CL.

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.

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.

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-17b (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.

Abstract.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≥1), E2 inactive (E-I: E/P<1, ≥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: ≥8.5 mm, stages I-III) or preovulatory follicles (POF: ≥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. battoir-derived ovaries have been used extensively as sources of oocytes for embryo production in vitro. Ovaries also provide useful materials for investigating the physiological functions of follicles. Although recent development of ultrasonography enables researchers to monitor development of follicles in real time, follicular samples derived from abattoir-harvested ovaries continue to be cheap and valuable materials for investigating the biochemical natures of follicular fluid (FF) and ovarian functions such as gene/ protein expression. For this reason, a simple and accurate method to determine the physiological status of follicles is necessary. To date, some follicular classification methods have been advocated. These methods are based on morphological, histological and biochemical criteria, but none of them are without faults.Histological examination employs the incidence of pycnosis in granulosa cell nuclei and the degree of granulosa/thecal integrity as the criteria [1]. Although this method is sensitive enough to detect a slight sign o...