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PubMed1989

Development of preovulatory follicles expected to form short-lived corpora lutea in beef cows.

Oestrus, expected to be followed by a short luteal phase, was induced in post-partum cows by weaning their calves at 35 days after parturition. Ovaries containing the first preovulatory follicles (Type F) formed after parturition were collected 3 h after the onset of oestrus. For comparison, preovulatory follicles (Type C) were collected 3 h after the onset of oestrus in normally cycling cows. The number of granulosa cells was determined and the concentrations of receptors for follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in granulosa cells and for LH in theca cells were measured. Concentrations of oestradiol-17 beta, testosterone, androstenedione and progesterone in follicular fluid were also measured. Type F follicles contained about twice the number of granulosa cells (based on DNA) as did Type C follicles (45.8 +/- 11.3 and 24.5 +/- 3.9 micrograms DNA/follicle, respectively; P less than 0.05) but these cells had fewer receptors for LH (0.13 +/- 0.02 vs 0.29 +/- 0.03 fmol/micrograms DNA; P less than 0.01) and FSH (0.61 +/- 0.08 vs 1.3 +/- 0.29 fmol/micrograms DNA; P less than 0.08) than did those from Type C follicles. Additionally, there were fewer receptors for LH in theca tissue from Type F than from Type C follicles (28.3 +/- 5.2 vs 51.3 +/- 6.1 fmol/follicle; P less than 0.01). Concentrations of oestradiol-17 beta (475.8 +/- 85.6 vs 112.9 +/- 40.0 ng/ml; P less than 0.01) and androstenedione (214.1 +/- 48.7 vs 24.7 +/- 7.7 ng/ml; P less than 0.01) in follicular fluid were higher in Type C than in Type F follicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Androstenedione↗
PubMed2006

The steroidogenic effect of single-chain bovine LH analogs in cultured bovine follicular cells.

Single-chain gonadotropin analogs had been constructed for the purpose of structure-function studies and analog design. Incorporation of a spacer derived from the carboxyl terminal peptide (CTP) of the choriogonadotropin (CG) beta subunit between the tethered subunit domains of the human gonadotropins is beneficial for the secretion of the single-chain variants without compromising biocactivity. Although the CGbeta subunit containing the CTP domain is expressed only in primates and equids, a CTP-like sequence exists in the untranslated region of the LHbeta gene of several mammalian species, including the bovine species. The CTP encrypted in the bovine LHbeta DNA (designated as 'boCTP') and the CTP derived from the human CGbeta subunit (denoted as 'huCTP') served as a linker sequence in the design of bovine single-chain luteinizing hormone (LH) analogs. The purpose of the present study was to evaluate steroidogenesis in cultured bovine theca cells following stimulation with these single-chain analogs. The concentration of the LHbetaboCTPalpha and LHbetahuCTPalpha analogs in the conditioned media of the expressing CHO cells was three- to six-fold higher than that of the "linkerless" LHbetaalpha and LHbeta111alpha variants. The four analogs induced androstenedione and progesterone secretion from the primary theca cells in a dose-dependent manner, but differences in the steroidogenic response were observed. The LHbetaboCTPalpha analog (10 ng/ml) effectively induced androstenedione and progesterone secretion over unstimulated levels (4.0- and 4.4-fold increase for androstenedione and progesterone, respectively). The response to the pituitary bovine LH standard (10 ng/ml) was less pronounced for both steroids (two- to three-fold increase over basal levels). The activities of LHbetahuCTPalpha, LHbetaalpha and LHbeta111alpha were comparable and sightly reduced relative to the LHbetaboCTPalpha activity. The data suggested that LHbetaboCTPalpha was ranked as the most potent and this was even more prominent when analogs were used at a lower dose (1 ng/ml). These data suggest that the design, including the huCTP or boCTP linker, is favorable for the production of single-chain bovine LH analogs. Furthermore, spacing of the tethered subunit domains with the cryptic boCTP sequence that originated from the bovine LHbeta gene appears advantageous for the purpose of stimulating steroid production in the species-specific bioassay. Thus, an effective strategy to produce bioactive single-chain LH analogs in non-primate, non-equid species would be the mutatation of the LHbeta genes with the aim of expressing the cryptic CTP sequence as a spacer derived from the DNA of the same organism.

Androstenedione↗
PubMed2003

Effect of insulin on rat ovarian leptin expression by immunohistochemical staining.

AIM: Leptin and insulin may interact in regulating ovarian steroid synthesis. The objective of this study was to investigate immunohistochemical staining of leptin in normal rat ovarian tissues and in rats treated with insulin and insulin plus human chorinoic gonadotropin (hCG). METHODS: Paraffin blocks of rat ovarian tissues from a previous study, in which 18 adult, female Wistar rats with an average weight of 250 g were divided into three groups to receive either saline solution, human insulin (2 U/day) or human insulin (2 U/day) plus hCG (4 U/day) for 4 weeks, were used in this study to compare the effects on leptin staining. The results were analysed using a semiquantitative scoring system, such as mild, moderate and strong. RESULTS: No staining was observed in granulosa cells and theca interna cells of normal ovarian tissues. Theca externa cells had mild staining intensity (+), corpus luteum had moderate (+ +) and stroma had mild (+) staining intensity. Histological structure was impaired in the insulin group, luteinized cells had mild staining, there was no difference in other cell groups. Only theca externa cells of the developing follicles were stained in insulin plus hCG group, luteinized cells again had mild staining. CONCLUSIONS: Besides damaging the rat ovarian structure, insulin reduced staining intensity of leptin in luteinized cells. Insulin may stimulate ovarian steroid synthesis not only through its own receptors, but also by acting on the leptin expression of these cells.

Animals↗
PubMed1995

Differential distribution of gangliosides in adult rat ovary during the oestrous cycle.

Gangliosides are ubiquitous membrane components in mammalian cells and are suggested to play important roles in various cell functions, such as cell-cell recognition, differentiation and transmembrane signalling. Rat ovary contained GM3, GD3 and GD1a as major gangliosides, and GM1 as a minor one. In order to study their distribution in the rat ovary and its possible changes during the oestrous cycle, frozen sections were stained with specific monoclonal antibodies against 11 ganglio-series gangliosides including those mentioned above. GM3, GM1 and GD1a were expressed in a spatio-temporally different manner during the oestrous cycle, but GD3 and other gangliosides were not immunohistochemically detected. In primary and secondary follicles, GM3, GM1 and GD1a were expressed in theca cells, but not in granulosa cells. The oocyte in primary, but not secondary, follicles was positive to the anti-GD1a antibody. In Graafian follicles, GM1 and GD1a were similarly expressed as in secondary follicles, however, the expression of GM3 spread gradually from theca cells to granulosa cells. In early Graafian follicles, only GM3 was expressed to a detectable extent from the outer part of the granulosa layer to the inner part. Shortly before ovulation, all granulosa cells and cumulus cells became positive to anti-GM3 antibody. After ovulation, differential distribution of GM3, GM1 and GD1a was also observed in luteal cells. GD1a was localized in thread-like structures, while GM3 was distributed throughout the cytoplasm, but not in the nucleus. GM1 was localized only in the plasma membrane and/or its close vicinity. Other ganglio-series gangliosides, including GD3, were not detected to an appreciable extent in the ovaries by immunohistochemistry.

Animals↗
PubMed1996

Elevated interleukin-6 levels in the ovarian hyperstimulation syndrome: ovarian immunohistochemical localization of interleukin-6 signal.

OBJECTIVE: To examine the production and immunolocalization of interleukin-6 (IL-6) in patients with the ovarian hyperstimulation syndrome. METHODS: The study group consisted of patients with ovarian hyperstimulation syndrome (n = 9) from whom serum and ascites samples were obtained. The control samples used were serum (n = 10) and peritoneal (n = 16) and follicular fluids (n = 8) from healthy individuals. Follicular fluid (n = 40) and serial serum samples were also obtained from patients undergoing menotropin stimulation for in vitro fertilization (IVF) before (n = 10) and after ovulation (n = 34). Interleukin-6 measurements were performed with a sensitive immunoassay and confirmed with a bioassay. Immunohistochemical localization of IL-6 was performed with a mouse monoclonal antibody in normal premenopausal (n = 5) and postmenopausal ovaries (n = 5), as well as with cells from stimulated follicular fluid aspirates (n = 3). RESULTS: We found significantly higher serum and ascites IL-6 levels in ovarian hyperstimulation syndrome (mean 18.8 +/- 1.1 and 810.8 +/- 60.7 pg/mL, respectively) compared with postovulatory serum and peritoneal fluid from normal controls (mean 4.4 +/- .69 and 44.7 +/- 7.5 pg/mL, respectively) (P < .001) or serum after menotropin stimulation (13.1 +/- 1.1 pg/mL) (P < .001). At the time of ovulation, follicular fluid IL-6 levels (normal controls, mean 9 +/- 2.1 pg/mL; menotropin stimulation, mean 10.1 +/- 4 pg/mL) were higher than in preovulatory serum (normal controls, mean 4.5 +/- .8 pg/mL; menotropin stimulation, mean 6.3 +/- 1.4 pg/mL) (P < .001). Immunohistochemical localization of IL-6 revealed intense staining in corpora lutea and theca cells from large antral follicles and luteinized granulosa cells in follicular aspirates after menotropin stimulation. CONCLUSION: Interleukin-6 levels are markedly elevated in the ovarian hyperstimulation syndrome when compared with controls. The higher follicular fluid IL-6 levels seen suggest local secretion of this cytokine. Immunohistochemical correlation demonstrated IL-6 within ovarian theca cells. These findings suggest a local role for IL-6 both in normal and stimulated ovarian function. Whether IL-6 is directly responsible for the clinical manifestations of this syndrome is unclear. However, when produced in massive amounts, the pro-inflammatory effects of IL-6 may contribute to its pathogenesis and perhaps serve as a marker for the disease.

Adult↗
PubMed2005

Steroidogenic activities of follicle-stimulating hormone in the ovary of Japanese eel, Anguilla japonica.

To clarify the physiological functions of follicle-stimulating hormone (FSH) during oogenesis in Japanese eel, Anguilla japonica, the steroidogenic activities of recombinant Japanese eel FSH (rjeFSH) were assessed in the eel ovary. Female eel were injected with salmon pituitary homogenate to enhance the ovarian development, and the ovaries at different developmental stages were subjected to steroidogenic bioassay. These ovaries could be classified into three types according to oocyte growth and development of ovarian follicular cells. The type-A ovary possessed poorly developed follicular cells around pre- or early vitellogenic oocytes, and rjeFSH did not induce sex steroid secretion. Testosterone (T) secretion was stimulated by rjeFSH in the type-B ovary with developed theca cells and undeveloped granulosa cells around early to mid-vitellogenic oocytes, whereas estradiol-17beta (E2) secretion was not enhanced. The rjeFSH stimulated both T and E2 secretion in a dose-dependent manner from the type-C ovary with fully developed theca and granulosa cells around mid-vitellogenic oocytes. Salmon GTH fraction (sGTH) and a membrane permeable cAMP analogue, 8-bromo-cAMP (8-Br-cAMP) also enhanced T and E2 secretion from the type-C ovary. Human chorionic gonadotropin (hCG) similarly enhanced T secretion, but failed to stimulate E2 secretion from the type-C ovary, suggesting different effects on steroidogenic activities between eel FSH and hCG in eel ovary. There was a positive correlation between the oocyte diameter and E2 secretion from eel ovaries stimulated by rjeFSH. These results suggest that aromatase activity is accelerated by eel FSH in the granulosa cells, which develop following theca cell development in this species.

Anguilla↗
PubMed1997

Alpha and beta subunits of inhibin/activin as sex cord-stromal differentiation markers.

Inhibins (alpha and beta heterodimers) and activins (beta homodimers) are related peptides with opposing biologic action on gonadotropin regulation. They serve as components of the pituitary-gonadal feedback system. Although sex-cord stromal tumors can usually be distinguished from ovarian epithelial tumors or their metastases by morphology or by using antibodies against intermediate filaments, the diagnosis remains difficult in rare situations in such cases as sarcomatoid granulosa-theca cell tumors, ovarian small cell carcinomas, or soft-tissue sarcomas. A total of 28 sex cord-stromal tumors of the ovary and 43 non-sex cord-stromal tumors were immunohistochemically evaluated for the presence of alpha and beta subunits of inhibin and activin. For comparison, 10 normal adult gonads including seven ovaries with hilar regions and three testes also were examined. Immunoreactivity for both alpha and beta subunits of inhibin/activin was identified in both non-neoplastic and neoplastic granulosa, Sertoli, Leydig, hilar and luteinized theca cells, with the strongest immunoreactivity in Leydig and hilar cells. One of three Sertoli-Leydig cell tumors that showed a sarcomatoid growth pattern and one sex-cord tumor with annular tubules also were immunoreactive for both subunits. For non-sex cord stromal-derived ovarian tumors, alpha subunit immunoreactivity was negative in all but two of five ovarian mucinous tumors. Weak immunoreactivity for beta subunit was found in most ovarian surface epithelial carcinomas, two of four colonic, and one of three pancreatic carcinomas. No immunostaining was found in nonspecialized gonadal stromal or interstitial cells, thecal cells, germ cells, ovarian small cell carcinomas, carcinoid tumors, dysgerminomas, or leiomyosarcomas. Immunostaining of alpha subunit (inhibin alpha), but not of beta subunit could serve as a sex cord-stromal differentiation marker because alpha subunit alone is largely confined to sex cord-stromal lesions with the exception of some ovarian mucinous tumors. Further studies are needed to define the usefulness of this sex cord-stromal differentiation marker in the practice of surgical pathology. Coexpression of alpha and beta subunits in sex cord-stromal elements suggests that dimeric inhibin is expressed in these cells.

Activins↗
PubMed2001

Basic fibroblast growth factor induces primordial follicle development and initiates folliculogenesis.

The recruitment of primordial follicles to initiate folliculogenesis determines the population of developing follicles available for ovulation and directly regulates female reproductive efficiency. In the current study, a floating organ culture system was used to examine the progression of primordial (stage 0) follicles to developing (stages 1-4) follicles in 4-day-old pre-pubertal rat ovaries. Basic fibroblast growth factor (bFGF) was found to induce primordial follicle development similar to what has been demonstrated for kit ligand/stem cell factor (KL). The bFGF-treated ovaries contained 85% developing follicles compared with 50% developing follicles for control untreated organ cultures. Correspondingly, the number of primordial follicles in bFGF-treated ovaries decreased to 15% of the total compared with 45% for controls. A bFGF neutralizing antibody was found to decrease the small amount of spontaneous follicle development that occurs during the organ culture. Basic FGF was localized to primordial and early developing follicles by immunocytochemistry and was primarily observed in the oocytes. Treatment of bovine ovarian theca cells and stroma cells with bFGF was found to promote cell growth. Basic FGF produced by the oocyte in early stage follicles appears to act on adjacent somatic cells to promote cell growth and development. Basic FGF, like KL, appears to be a primordial follicle-inducing factor. In summary, bFGF can regulate primordial follicle development that directly influences female reproductive efficiency.

Animals↗
PubMed1986

Bovine theca and granulosa cells interact to promote androgen production.

Pieces of theca interna or follicle wall (theca interna + attached granulosa cells), obtained from bovine preovulatory follicles prior to the surge of luteinizing hormone (LH) and cultured for 3 days, secreted androstenedione. Luteinizing hormone, but not follicle-stimulating hormone (FSH), increased production of androstenedione 3 to 4-fold. In both the presence and absence of LH, follicle wall preparations secreted about 4-fold more androstenedione than did equivalent amounts of theca interna tissue. Isolated granulosa cells produced only negligible quantities of androstenedione, which suggests that they may contribute to the greater production of androstenedione by follicle wall by supplying progestin precursor to the theca cells. The addition of pregnenolone or progesterone to isolated theca interna increased the secretion of androstenedione, but pregnenolone was by far the more effective precursor. This suggested that the delta 5 (delta 5) pathway is the preferred pathway for androstenedione synthesis by bovine theca cells and that granulosa cells might supply progestin precursor in the form of pregnenolone. Follicle wall and granulosa cell cultures secreted 2 and 7 times more pregnenolone, respectively, than did theca cultures. Luteinizing hormone, but not FSH, increased production of pregnenolone by the follicle wall, whereas the gonadotropins had no effect on secretion by either granulosa or theca cells. Since exogenous testosterone enhanced the production of pregnenolone by granulosa cells, thecal androgen (which is stimulated by LH) may increase the ability of granulosa cells to make pregnenolone and explain the stimulatory effect of LH on pregnenolone secretion by follicle wall.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgens↗
PubMed1993

Oxytocin gene expression and action in bovine preovulatory follicles.

We have studied oxytocin (OT) gene expression, secretion, and action in bovine preovulatory follicles during the follicular phase of the estrous cycle. OT is secreted in vitro by follicular granulosa cells, but not by theca cells. Both OT content of granulosa cells and their ability to secrete OT in culture increased dramatically when follicles were obtained after the gonadotropin surge (LH surge) that triggers ovulation. These changes were correlated with increased levels of messenger RNA (mRNA) for OT in granulosa cells obtained after vs. before the LH surge. When granulosa cells were obtained before the surge, both OT secretion and OT mRNA levels increased with time in culture, and the increases were greatly enhanced in the presence of LH. Estradiol, at concentrations found in follicular fluid of preovulatory follicles before the LH surge, inhibited OT secretion in vitro, whereas concentrations found in follicular fluid after the LH surge were not inhibitory. Progesterone, at physiological concentrations, stimulated OT secretion in vitro. We have shown previously that OT increases progesterone secretion by granulosa cells obtained before the LH surge. Taken together these results show that, during the follicular phase in cattle, OT secretion and gene expression are coordinately regulated and suggest that they are regulated by both gonadotropins and intrafollicular steroids. Increases in OT after the LH surge may play a role in the follicular/luteal phase shift in steroidogenesis from estradiol/androgen to progesterone.

Animals↗
PubMed2005

Polychlorinated biphenyls (PCB 153 and PCB 126) action on conversion of 20-hydroxylated cholesterol to progesterone, androstenedione to testosterone, and testosterone to estradiol 17beta.

To look for one of the possible mechanisms of action we investigated the effect of two congeners of polychlorinated biphenyls (PCB153 as one of the most prominent environmental contaminants and PCB 126 as one of the most toxic contaminants similar to dioxin) on the cellular conversion of steroid precursors as an indicator for enzyme activity (20-hydroxylated cholesterol to progesterone for P450 (scc,) androstendione to testosterone for 17-beta-HSD, and testosterone to estradiol for P450 (arom)). The net synthesis and secretion of particular steroids was used as the indicator of enzyme activity. Co-culture of pig granulosa and theca cells isolated from small (SF) and large (LF) follicles, was carried out in medium M199 supplemented with 100 ng/ml of PCB 153 or 100 pg/ml of PCB 126. The inhibitory action of both PCB 126 and PCB 153 on progesterone secretion by cells isolated from SF and LF follicles was reversed in the presence of 20-hydroxylated cholesterol. The addition of PCB 126 into the culture medium caused a decrease in testosterone secretion by cells isolated from both SF and LF and this effect was reversed in the presence of androstendione. The inhibitory action of PCB 153 on testosterone secretion was reversed by the addition of androstendione to the culture medium in SF, while it caused even additional stimulatory action on cells collected from LF. No effect of PCB 126 and statistically significant decrease in estradiol secretion by cells collected from SF under the influence of PCB153 was observed. The inhibitory effect of PCB 153 was reversed when the culture was supplemented with testosterone. The opposite effect of both tested congeners on estradiol secretion in both basal and testosterone supplemented culture was seen in LF. PCB 126 increased it while PCB 153 decreased both, the basal and testosterone-stimulated estradiol secretion. In conclusion, the presented results suggest that the effect of both PCBs on steroid secretion observed in an early stage of the follicular phase of the estrus cycle is due to the inhibition of cholesterol mobilisation and thus insufficient substrate availability for hormone synthesis. On the contrary, in large preovulatory follicles inhibition of testosterone secretion is due to their action on 17-beta-HSD while stimulatory or inhibitory action on estradiol secretion is the result of their action on P450 aromatase activity.

Animals↗
PubMed1998

Oxysterol regulation of steroidogenic acute regulatory protein gene expression. Structural specificity and transcriptional and posttranscriptional actions.

Oxysterols exert a major influence over cellular cholesterol homeostasis. We examined the effects of oxysterols on the expression of steroidogenic acute regulatory protein (StAR), which increases the delivery of cholesterol to sterol-metabolizing P450s in the mitochondria. 22(R)-hydroxycholesterol (22(R)-OHC), 25-OHC, and 27-OHC each increased steroidogenic factor-1 (SF-1)-mediated StAR gene transactivation by approximately 2-fold in CV-1 cells. In contrast, cholesterol, progesterone, and the 27-OHC metabolites, 27-OHC-5beta-3-one and 7alpha,27-OHC, had no effect. Unlike our findings in CV-1 cells, SF-1-dependent StAR promoter activity was not augmented by 27-OHC in COS-1 cells, Y-1 cells, BeWo choriocarcinoma cells, Chinese hamster ovary (CHO) cells, and human granulosa cells. Studies examining the metabolism of 27-OHC indicated that CV-1 cells formed a single polar metabolite, 3beta-OH-5-cholestenoic acid from radiolabeled 27-OHC. However, this metabolite inhibited StAR promoter activity in CV-1, COS-1 and CHO cells. Because 7alpha,27-OHC was unable to increase SF-1-dependent StAR promoter activity, we examined 27-OHC 7alpha-hydroxylase in COS-1 and CHO cells. COS-1 cells contained high 7alpha-hydroxylase activity, whereas the enzyme was undetectable in CHO cells. The hypothesis that oxysterols act in CV-1 cells to increase StAR promoter activity by reducing nuclear levels of sterol regulatory element binding protein was tested. This notion was refuted when it was discovered that sterol regulatory element binding protein-1a is a potent activator of the StAR promoter in CV-1, COS-1, and human granulosa cells. Human granulosa and theca cells, which express endogenous SF-1, contained more than 5-fold more StAR protein following addition of 27-OHC, whereas StAR mRNA levels remained unchanged. We conclude that 1) there are cell-specific effects of oxysterols on SF-1-dependent transactivation; 2) the ability to increase transactivation is limited to certain oxysterols; 3) there are cell-specific pathways of oxysterol metabolism; and 4) oxysterols elevate StAR protein levels through posttranscriptional actions.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2004

Mouse very long-chain Acyl-CoA synthetase 3/fatty acid transport protein 3 catalyzes fatty acid activation but not fatty acid transport in MA-10 cells.

The family of proteins that includes very long-chain acyl-CoA synthetases (ACSVL) consists of six members. These enzymes have also been designated fatty acid transport proteins. We cloned full-length mouse Acsvl3 cDNA and characterized its protein product ACSVL3/fatty acid transport protein 3. The predicted amino acid sequence contains two highly conserved motifs characteristic of acyl-CoA synthetases. Northern blot analysis revealed that the mouse Acsvl3 mRNA is highly expressed in adrenal gland, testis, and ovary, with lower expression in the brain of adult mice. A developmental Northern blot revealed that Acsvl3 mRNA levels were significantly higher in embryonic mouse brain (embryonic days 12-14) than in newborn or adult mice, suggesting a possible role in nervous system development. Immunohistochemistry revealed high ACSVL3 expression in adrenal cortical cells, spermatocytes and interstitial cells of the testis, theca cells of the ovary, cerebral cortical neurons, and cerebellar Purkinje cells. Endogenous ACSVL3 was found primarily in mitochondria of MA-10 and Neuro2a cells by both Western blot analysis of subcellular fractions and immunofluorescence analysis. In MA-10 cells, loss-of-function studies using RNA interference confirmed that endogenous ACSVL3 is an acyl-CoA synthetase capable of activating both long-chain (C16:0) and very long-chain (C24:0) fatty acids. However, despite decreased acyl-CoA synthetase activity, initial rates of fatty acid uptake were unaffected by knockdown of Acsvl3 expression in MA-10 cells. These studies cast doubt on the designation of ACSVL3 as a fatty acid transport protein.

Adrenal Glands↗