Activin effects on follicular growth in in vitro preantral follicle culture.

As the follicular environment transits from being activin dominant to inhibin dominant during folliculogenesis, it is assumed that activin plays an important role in the early stage of follicular growth. We examined the effects of activin on morphological, biochemical and molecular changes in isolated preantral follicles. Preantral follicles were mechanically isolated from 14-day old female C57BL/6 mice. Each follicle was cultured and observed for 14 days usingan in vitro follicle culture system containing FSH, FSH + activin A and FSH + inhibin in the culture medium. We subsequently examined FSH receptor (FSH-R) mRNA expression in isolated follicle cultures with or without activin on days 0 and 2. Activin was observed to significantly stimulate follicle enlargement on days 2, 4, 6 and 8, accelerate morphological changes and increase estradiollevels in culture medium on days 4, 12 and 14. In contrast, inhibin did not alter follicular growth. Additionally, activin stimulated the expression of FSH-R mRNA in isolated granulosa cells. It was demonstrated that activin stimulated the growth of preantral follicles, mainly during the early stage of folliculogenesis, by inducing FSH-R expression, in an isolated follicle culture system. J. Med. Invest. 66 : 165-171, February, 2019.

Methods for Evaluation of Ovarian Granulosa Cells with Exposure to Nanoparticles.

Increasing evidence demonstrates that a substantial number of chemicals including nanoparticles have potential toxic effects on human and wildlife reproductive health. Ovarian granulosa cells (GCs), the functional unit of the ovary, play a pivotal role in the regulation of ovarian reproductive health in females. They are considered to be the main target by the chemicals at all stages of their development. It has been reported that nanoparticles induced toxicity of GCs by interfering with cell cycle. Some basic methods for evaluation of GCs with exposure to nanoparticles are described in this chapter including isolation of GCs, determination of apoptosis by immunofluorescence, and measurement of progesterone level by ELISA.

Expression Levels of Follicle-Stimulating Hormone Receptor and Implication in Diagnostic and Therapeutic Strategy of Ovarian Cancer.

BACKGROUND: Follicle-stimulating hormone receptor (FSHR) has been shown to be expressed in ovarian cancer. METHODS: Here we have summarized the potential therapeutic and diagnostic implication of FSHR in the ovarian cancers based on a review of the literature. RESULTS: Current research indicates that FSHR comprises several variants: FSHR-1, FSHR-2, FSHR-3 and FSHR-4. Only FSHR-1 and FSHR-3 have biological roles. Although the level of FSHR differs in ovarian cancer tissues, few quantitative correlations have so far been reported on the expression levels of FSHR and carcinogenesis and progression of cancers. CONCLUSION: A comprehensive understanding of the role of FSHR in the ovarian cancers may help the search for novel therapeutic and diagnostic regimens and improve the management of cancer patients.

PET Imaging of FSHR Expression in Tumors with 68Ga-Labeled FSH1 Peptide.

FSHR is an appealing target for cancer theranostics. Radiolabeled FSH1 and its derivatives have shown potential to in vivo detect FSHR expression. However, moderate labeling yields (~50% nondecay-corrected) may partially limit their wide use. 68Ga is an excellent PET nuclide due to availability, nearly quantitative reaction, and short physical half-life. In this study, 68Ga labeled FSH1 peptide was developed for imaging of FSHR in cancers. In vitro studies and MicroPET imaging were performed in PC-3 prostate tumor model. [68Ga] Ga-NOTA-MAL-FSH1 can be produced within 20 min with 93.2 ± 2.1% yield and the radiochemical purity was greater than 95%. It showed that [68Ga] Ga-NOTA-MAL-FSH1 possessed FSHR binding affinities. The tracer was stable in PBS and human serum for at least 2 hours. MicroPET imaging revealed that the PC-3 xenografts were clearly visualized and the tumor uptakes were 1.87 ± 0.10, 1.26 ± 0.06, and 0.71 ± 0.10% ID/g at 0.5, 1 h, and 2 h postinjection. The corresponding tumor to blood and tumor to muscle ratios were 1.77 ± 0.70, 7.94 ± 1.35, and 10.37 ± 1.16 and 7.42 ± 0.46, 26.13 ± 2.99, and 36.40 ± 2.54, respectively. FSHR binding specificity was also demonstrated by reduced tumor uptake of [68Ga] Ga-NOTA-MAL-FSH1 after coinjecting excess unlabeled FSH1 peptide. The favorable characters of [68Ga] Ga-NOTA-MAL-FSH1 such as convenient synthesis and specific tumor uptake warrant its further investigation for FSHR expression imaging.

A differential cytokine expression profile before and after rFSH treatment in Sertoli cell cultures of men with nonobstructive azoospermia.

To evaluate the effects of follicle-stimulating hormone (FSH) treatment on cytokine gene expression in cultured Sertoli cells from men with nonobstructive azoospermia, a total of 15 azoospermic men diagnosed as obstructive azoospermia (OA) (n = 5) and nonobstructive azoospermia (NOA) (n = 10) were included in the study. NOA patients were split into two further subgroups: nFSH and hFSH serum FSH levels. Expression of cytokine gene panel (88 genes), FSHR and ABP was evaluated by real-time PCR array analysis. FSHR protein level was measured by the Western blot. In primary cultures of Sertoli cells, seven genes were found to be increased and 13 were decreased in NOA group, when compared to OA (p < .05). When rFSH was introduced into the culture media, expression of 12 genes in the NOA group restored a comparable level to those of the control OA group. Sertoli cells in all groups responded rFSH administration with increased expression of ABP. Our results suggest that FSH treatment may have positive effects on Sertoli cells of nonobstructive azoospermic patients via changing the expression levels of certain genes and restoring their levels in normal Sertoli cell population. Some cytokine levels can be considered as a potential candidate for detecting NOA patients. ABP is a good marker for cell viability and functionality in primary Sertoli cell culture.

FSH aggravates bone loss in ovariectomised rats with experimental periapical periodontitis.

Periapical bone loss is one of the prominent pathological and clinical features of periapical periodontitis. Previous studies have demonstrated that follicle­stimulating hormone (FSH) could directly affect skeletal remodelling by stimulating the formation and the function of osteoclasts in vitro and in vivo. However, the effect of FSH on periapical bone loss remained to be fully elucidated. In the current study, a rat model was established in order to verify the effect of FSH in experimental periapical lesions. It was identified that FSH aggravated the bone loss of periapical lesions. In addition, RANKL­, TRAP­, TNF­α­ and IL­1ß­positive cells were increased significantly in FSH­treated groups, which indicated that the function of FSH in bone loss may be mediated through the increasing activity of osteoclasts and the increased secretion of inflammatory cytokines. The results of the current study suggested that FSH, independent of oestrogen, may aggravate periapical bone loss by FSH receptors, which may serve an important role in the immune and inflammatory response of the host to root canal and periradicular infection during menopause.

Kisspeptin is involved in ovarian follicular development during aging in rats.

We have previously reported that kisspeptin (KP) may be under the control of the sympathetic innervation of the ovary. Considering that the sympathetic activity of the ovary increases with aging, it is possible that ovarian KP also increases during this period and participates in follicular development. To evaluate this possibility, we determined ovarian KP expression and its action on follicular development during reproductive aging in rats. We measured ovarian KP mRNA and protein levels in 6-, 8-, 10- and 12-month-old rats. To evaluate follicular developmental changes, intraovarian administration of KP or its antagonist, peptide 234 (P234), was performed using a mini-osmotic pump, and to evaluate FSH receptor (FSHR) changes in the senescent ovary, we stimulated cultured ovaries with KP, P234 and isoproterenol (ISO). Our results shows that KP expression in the ovary was increased in 10- and 12-month-old rats compared with 6-month-old rats, and this increase in KP was strongly correlated with the increase in ovarian norepinephrine observed with aging. The administration of KP produced an increase in corpora lutea and type III follicles in 6- and 10-month-old rats, which was reversed by P234 administration at 10 months. In addition, KP decreased the number and size of antral follicles in 6- and 10-month-old rats, while P234 administration produced an increase in these structures at the same ages. In ovarian cultures KP prevented the induction of FSHR by ISO. These results suggest that intraovarian KP negatively participates in the acquisition of FSHR, indicating a local role in the regulation of follicular development and ovulation during reproductive aging.

Mechanisms Underlying Testicular Damage and Dysfunction in Mice With Partial IGF-1 Deficiency and the Effectiveness of IGF-1 Replacement Therapy.

OBJECTIVE: To determine whether insulin-like growth factor (IGF-1) deficiency can cause testicular damage and to examine changes of the testicular morphology and testicular function-related gene expression caused by IGF-1 deficiency. Therefore, this study aims to determine the benefits of low doses of IGF-1 and to explore the mechanisms underlying the IGF-1 replacement therapy. MATERIALS AND METHODS: A murine model of IGF-1 deficiency was used to avoid any factor that could contribute to testicular damage. Testicular weight, score of histopathological damage, and gene expressions were studied in 3 experimental groups of mice: controls (wild-type Igf1(+/+)), heterozygous Igf1(+/-) with partial IGF-1 deficiency, and heterozygous Igf1(+/-) treated with IGF-1. RESULTS: Results show that the partial IGF-1 deficiency induced testicular damage and altered expression of genes involved in IGF-1 and growth hormone signaling and regulation, testicular hormonal function, extracellular matrix establishment and its regulation, angiogenesis, fibrogenesis, inflammation, and cytoprotection. In addition, proteins involved in tight junction expression were found to be reduced. However, low doses of IGF-1 restored the testicular damage and most of these parameters. CONCLUSION: IGF-1 deficiency caused the damage of the blood-testis barrier and testicular structure and induced the abnormal testicular function-related gene expressions. However, low doses of IGF-1 constitute an effective replacement therapy that restores the described testicular damage. Data herein show that (1) cytoprotective activities of IGF-1 seem to be mediated by heat shock proteins and that (2) connective tissue growth factor could play a relevant role together with IGF-1 in the extracellular matrix establishment.

Serum levels of sex hormones and expression of their receptors in thyroid tissue in female patients with various types of thyroid neoplasms.

Previous studies have demonstrated the expression of estrogen receptor (ER) and progesterone receptor (PR) in thyroid cancer; however, little is known regarding the levels of estrogen, progesterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) in serum and the expression of ER, PR, FSH receptor (FSHR), and LH receptor (LHR) in thyroid tissues of patients with different types of thyroid neoplasms. Serum levels of estrogen, progesterone, FSH, and LH were measured by chemiluminescence, and expression of ER, PR, FSHR, and LHR in thyroid tissue was detected by immunohistochemistry in female patients with thyroid adenoma (n = 70), nodular goiter (n = 73), thyroid papillary cancer (n = 149), poorly differentiated thyroid carcinoma (n = 12), or undifferentiated thyroid carcinoma (n = 8) and in normal controls (n = 60). The positive rates of serum estrogen level and ERα expression were significantly greater in patients with various types of thyroid neoplasms than in normal controls. The positive rates of ERß expression were significantly less in various types of thyroid neoplasms than in normal thyroid tissues, especially in poorly differentiated carcinoma and undifferentiated carcinoma. The negative rates of serum progesterone level and positive rates of PR expression in thyroid tissue were significantly greater in patients with thyroid adenoma, nodular goiter, or thyroid papillary cancer than in normal controls. The positive rates of serum FSH and LH levels and FSHR and LHR expression were significantly greater in the thyroid adenoma group than in other groups. Our findings suggest that thyroid neoplasms might be sex hormone-dependent. The positive expression of ERα and PR often indicates thyroid papillary carcinoma, and the ERß expression status is important for the diagnosis of poorly differentiated carcinoma and undifferentiated carcinoma. In addition, thyroid adenoma is often accompanied by an increase in serum FSH and LH levels, as well as FSHR and LHR expression. Thus, the combined detection of serum levels of sex hormones and expression of their receptors allows for a differential diagnosis and evaluation of the degree of differentiation among various types of thyroid neoplasms.

Immunolocalization of GnRHRI, gonadotropin receptors, PGR, and PGRMCI during follicular development in the rabbit ovary.

The aim of this study was to investigate the presence and localization of gonadotropin-releasing hormone receptor-I (GnRHRI), gonadotropin receptors (FSHR, LHR), progesterone receptor (PGR), and progesterone receptor membrane-binding component-I (PGRMCI) in the different developmental stages of the rabbit follicle. The ovaries were collected from four healthy New Zealand white rabbits, and the mRNA expression and protein levels of GnRHRI, FSHR, LHR, PGR, and PGRMCI were examined with real-time PCR and immunohistochemistry. The results showed that GnRHRI, FSHR, LHR, PGR, and PGRMCI mRNA was expressed in the ovary; furthermore, we show cell-type specific and follicular development stage-specific expression of these receptors at the protein level. Specifically, all of the receptors were detected in the oocytes from the primordial to the tertiary follicles and in the granulosa and theca cells from the secondary and tertiary follicles. In the mature follicles, all receptors were primarily localized in the granulosa and theca cells. In addition, LHR was also localized in the granulosa cells from the primordial and primary follicles. With follicular development, the expression level of all of the receptors, except GnRHRI, in the follicles showed a tendency to decrease because the area of the follicle increased sharply. The expression level of GnRHRI, FSHR, and PGR in the granulosa and theca cells showed an increasing trend with ongoing follicular development. Interestingly, the expression level of FSHR in the oocytes obviously decreased from the primary to the tertiary follicles, whereas LHR in the oocytes increased from the secondary to tertiary follicles. In conclusion, the expression of GnRHRI, the gonadotropin receptors, PGR, and PGRMCI decreased from the preantral follicles (primordial, primary, and secondary follicles) to the tertiary follicles. The expression of GnRHRI and LHR in the oocytes increased from the secondary to the tertiary follicles, whereas FSHR decreased from the primary to the tertiary follicles. The expression of GnRHRI and PGR in the granulosa and theca cells increased from the secondary to the mature follicles. These observations suggest that these receptors play roles in follicular development and participate in the regulation of follicular development.

Effects of flavonoids from semen cuscutae on the hippocampal-hypothalamic-pituitary-ovarian sex hormone receptors in female rats exposed to psychological stress.

OBJECTIVE: To investigate the effects of flavonoids from semen cuscutae (FSCs) on the hippocampal-hypothalamic-pituitary-ovarian sex hormone receptors in female rats exposed to psychological stress and to explore the related mechanism. MATERIALS AND METHODS: Flavonoids were obtained from semen cuscutae using solvent extraction and polyamide column chromatography. Sound, light, and electricity were combined into psychological stress for endocrine dysfunction model establishment in female rats. The effects of FSCs on estrogen receptor (ER) in the hippocampus, hypothalamus, and pituitaries, as well as on follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) in the ovaries of the psychologically stressed rats were quantitatively analyzed using immunohistochemistry and image analysis. RESULTS: FSCs increased ER expression in the hippocampus, hypothalamus, and pituitaries, as well as LHR expression in the ovaries, but had no effect on FSHR expression in the ovaries. CONCLUSION: FSCs are an effective medicine in the treatment of ovarian endocrine dysfunction in psychologically stressed rats.

Alarelin active immunization influences expression levels of GnRHR, FSHR and LHR proteins in the ovary and enhances follicular development in ewes.

We investigated the effects of gonadotropin releasing hormone (GnRH) agonist on expressions of GnRH receptor (GnRHR), follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) proteins in the ovaries and follicular development in the ewes. Forty-two pre-pubertal ewes were assigned to experimental groups 1 to 5 (EG-I to EG-V) and control group (CG). Ewes in EG-I, EG-II and EG-III were subcutaneously injected with 200, 300 or 400 µg alarelin antigens twice (on days 0 and 14), respectively. Ewes in EG-IV and EG-V were subcutaneously injected with 200 µg and 300 µg alarelin antigen four times (on days 0, 7, 14 and 21). Ewes in CG were subcutaneously injected with a solvent twice (on days 0 and 14). Serum concentrations of GnRH antibody in the EGs increased and were higher than (P<0.05) that of CG from day 14 to day 60. GnRH antibody concentrations in EG-IV and EG-V were higher than that in EG-I, EG-II and EG-III from days 35 to 45. Expressions of GnRHR protein in EG-IV and EG-V were lower than that in CG (P<0. 01). Expressions of FSHR and LHR proteins in EGs increased. Levels of FSHR and LHR proteins in EG-IV and EG-V (P<0.05) were higher than CG. Ovarian weights in EGs increased. Values of follicle vertical diameter, follicle transverse diameter, follicle wall thickness, follicle externatheca thickness and follicle internatheca thickness in EG-III and EG-V were greater than other groups. Primordial follicles and primary follicles developed quickly in alarelin-immunized animals. Secondary follicles and mature follicles became more abundant. Mitochondria, mitochondrial cristaes and cortical granules increased. Serum FSH concentrations of EGs remained higher than that in CG from days 28 to 70 (P<0.05). Alarelin immunization stimulated GnRH antibody production, suppressed expression of GnRHR protein, enhanced expressions of FSHR and LHR proteins in ovaries, promoted FSH secretion and thereby accelerated the development of ovaries and follicles in ewes.

Effect of FSH receptor-binding inhibitor-8 on FSH-mediated granulosa cell signaling and proliferation.

Follicle-stimulating hormone is important for mammalian reproduction. It acts through specific receptors located on the plasma membrane of granulosa cells in ovaries and Sertoli cells in testes. The binding of follicle-stimulating hormone to its receptor activates intracytoplasmic signaling pathways leading to steroidogenesis. These steroids in turn regulate the follicle-stimulating hormone action from the anterior pituitary through exerting negative feedback effect. In addition to steroids, non-steroidal factors secreted by the ovaries are believed to modulate follicle-stimulating hormone action through autocrine/paracrine mode. One such low molecular weight peptide referred to as follicle-stimulating hormone receptor-binding inhibitor-8 purified from human follicular fluid has been extensively studied. Follicle-stimulating hormone receptor-binding inhibitor-8 has been shown to inhibit binding of follicle-stimulating hormone to its receptor. The present article describes the effect of follicle-stimulating hormone receptor-binding inhibitor-8 on follicle-stimulating hormone-induced signaling in rat granulosa cells. Follicle-stimulating hormone receptor-binding inhibitor-8 inhibited the follicle-stimulating hormone-induced cAMP, and the effect was observed to be mediated through the protein kinase A. Further, an inhibitory effect of follicle-stimulating hormone receptor-binding inhibitor-8 on the granulosa cell proliferation was evaluated using COV434 cell line which is derived from the human granulosa cell tumor. The effect of the peptide on the cell cycle analysis showed an increase in apoptotic population and the arrest of G1 phase. These findings suggest that follicle-stimulating hormone receptor-binding inhibitor-8 acts as a follicle-stimulating hormone antagonist and affects the follicle-stimulating hormone-mediated signaling and proliferation in the granulosa cells.

Trafficking of the follitropin receptor.

The follitropin or follicle-stimulating hormone receptor (FSHR) belongs to a highly conserved subfamily of the G protein-coupled receptor (GPCR) superfamily and is mainly expressed in specific cells in the gonads. As any other GPCR, the newly synthesized FSHR has to be correctly folded and processed in order to traffic to the cell surface plasma membrane and interact with its cognate ligand. In this chapter, we describe in detail the conditions and procedures used to study outward trafficking of the FSHR from the endoplasmic reticulum to the plasma membrane. We also describe some methods to analyze phosphorylation, ß-arrestin recruitment, internalization, and recycling of this particular receptor, which have proved useful in our hands for dissecting its downward trafficking and fate following agonist stimulation.

Modern methods to investigate the oligomerization of glycoprotein hormone receptors (TSHR, LHR, FSHR).

As for other GPCRs, the oligomerization of glycoprotein hormone receptors (GPHRs) appears as critical event for receptor function. By means of modern techniques based on the BRET or FRET principle, GPHR oligomerization has been reported to explain several physiological and pathological conditions. In particular, the presence of oligomers was demonstrated not only in in vitro heterologous systems but also in in vivo tissues, and GPHR homodimerization appears associated with strong negative cooperativity, thus suggesting that one hormone molecule may be sufficient for receptor dimer stimulation. In addition, oligomerization has been reported to occur early during the posttranslational maturation process and to be involved in the dominant negative effect exerted by loss-of-function TSH receptor (TSHR) mutants, that are prevalently retained inside the cell, on the surface expression of wild-type receptors. This molecular mechanism thus explains the dominant inheritance of certain forms of TSH resistance. Here, we provide the description of the methods used in the original BRET, FRET, and HTRF-RET experiments.

Expression patterns of gonadotropin hormones and their receptors during early sexual differentiation in Nile tilapia Oreochromis niloticus.

In Nile tilapia, sex-specific expression of foxl2 and cyp19a1a in XX gonads and dmrt1 in XY gonads at 5-6 days after hatching (dah) is critical for differentiation of the gonads into either ovaries or testes. The factors triggering sexually dimorphic expression of these genes are unknown, and whether the gonadotropin hormones are involved in early gonadal sex differentiation of the Nile tilapia has been unclear. In the present study, we determined the precise timing of expression of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the pituitary and that of their receptors (fshra and lhcgrbb) in the undifferentiated gonad in both XX and XY tilapia fry by quantitative RT-PCR and immunohistochemical analysis. Expression of fshb mRNA and Fsh protein in the pituitary was detected from the first sampling day (3 dah) to 25 dah in both XX and XY tilapia larvae without sexual dimorphism and increased gradually after 25 dah in the pituitary. fshra mRNA was expressed beginning 5 dah and was present at significantly higher levels in XX gonads than in the XY gonads at 6-25 dah. These results indicate that the level of Fsh protein in the pituitary was not critical for differentiation of gonads into ovaries or testes, but the expression level of its receptor, fshra, in undifferentiated gonads appeared to be involved in determining gonadal sexual differentiation. Based on these observations, it is likely that in XX gonads, up-regulation of fshra may be necessary to induce cyp19a1a expression, which stimulates estradiol-17beta (E(2)) production and subsequent ovarian differentiation. On the other hand, lhb mRNA was not detected until 25 dah in the pituitaries of both sexes, and sexual dimorphism in lhcgrbb mRNA levels appeared later (10-25 dah) than that of fshra in the gonads, indicating the limited role of LH and lhcgrbb in gonadal differentiation of the Nile tilapia.

A switch in Sertoli cell responsiveness to FSH may be responsible for robust onset of germ cell differentiation during prepubartal testicular maturation in rats.

FSH and Testosterone (T) regulate spermatogenesis via testicular Sertoli cells (Sc), which bear receptors for these hormones. Despite sufficient circulating levels of FSH and T postnatally, predominant appearance of spermatogonia B and spermatocytes is not discernible until 11 and 18 days of postnatal age, respectively, in rat testes. In an attempt to explore the underlying causes, we cultured Sc from neonatal (5- and 9-day-old) and prepubertal (12- and 19-day-old) rat testes and compared the status of FSH receptor (FSH-R) and androgen receptor (AR) signaling. Protein and mRNA levels of FSH-R and AR remained uniform in cultured Sc from all age groups. Androgen binding ability of AR was similar, and T-induced nuclear localization of AR was discernible in Sc from all age groups. Binding of FSH to FSH-R, subsequent production of cAMP, and mRNA of stem cell factor (SCF) and glial cell line-derived neurotrophic factor (GDNF), known to be essential for the robust differentiation of repopulating spermatogonia, were significantly augmented in prepubertal Sc compared with those in neonatal Sc. However, treatment of neonatal Sc with cholera toxin or forskolin, which stimulate cAMP production bypassing FSH-R, demonstrated a concomitant rise in SCF and GDNF mRNA expression, which was similar to the FSH-mediated rise observed in prepubertal Sc. These observations suggested that, during prepubertal Sc maturation, the ability of FSH-R to respond to FSH is significantly augmented and is associated with the robust differentiation of repopulating spermatogonia, and such a switch in Sc from FSH-resistant to FSH-responsive mode during prepubertal development may underlie the initiation of robust spermatogenesis.

The effects of insulin and follicle-simulating hormone (FSH) during in vitro development of ovarian goat preantral follicles and the relative mRNA expression for insulin and FSH receptors and cytochrome P450 aromatase in cultured follicles.

The actions of different concentrations of insulin alone or in combination with follicle-stimulating hormone (FSH) were evaluated by in vitro follicular development and mRNA expression of cytochrome P450 aromatase (CYP19A1) and as receptors for insulin (INSR) and FSH (FSHR) from isolated, cultured goat preantral follicles. Goat preantral follicles were microdissected and cultured for 18 days in the absence or presence of insulin (5 and 10 ng/ml or 10 µg/ml) alone or in combination with FSH. After 18 days, the addition of the maximum concentration of insulin to the culture medium reduced follicular survival and antrum formation rates significantly compared to the other treatments. However, when FSH was added to the culture medium, no differences between these two parameters were observed. Preantral and antral follicles from the fresh control as well as from all cultured follicles still presented a normal ultrastructural pattern. In medium supplemented with FSH, only insulin at 10 ng/ml presented oocytes with higher rates of meiosis resumption compared to control, as well as oocytes in metaphase II. Treatment with insulin (10 ng/ml) plus FSH resulted in significantly increased levels of INSR and CYP19A1 mRNA compared to that with other treatments. In conclusion, 10 ng/ml insulin associated with FSH was more efficient in promoting resumption of oocyte meiosis, maintaining survival, stimulating follicular development, and increasing expression of the INSR and CYP19A1 genes in goat preantral follicles.

Endothelial follicle stimulating hormone receptor in primary kidney cancer correlates with subsequent response to sunitinib.

Sunitinib is an anti-angiogenic receptor tyrosine kinase inhibitor used to treat advanced metastatic renal cell carcinoma and other types of cancer. Sutent is effective in only approximately 70% of clear cell renal cell carcinoma (CCRCC) patients, has significant adverse side effects and no method is available to predict which patients will not respond. Our purpose was to explore the possibility of introducing an effective prediction method based on a marker of the tumour vasculature, the follicle stimulating hormone receptor (FSHR). Fifty patients diagnosed with advanced metastatic CCRCC have been subjected to surgery for removal of the primary tumour and were subsequently treated with sunitinib. After three months of therapy the patients were categorized as 'responsive', 'stable' or 'non-responsive' based on the RECIST guidelines. The blood vessel density and the percentage of FSHR-positive vessels were determined by immunofluorescence on sections from the primary tumours removed by surgery, prior to the sunitinib treatment. The percentage of FSHR-stained vessels was on average fivefold higher for the patients who responded to the treatment in comparison with the stable group and almost eightfold higher than in the non-responsive group. The percentage allowed the detection of responders with 87-100% sensitivity and specificity. No significant differences were detected in the total density of vessels among the three groups. The data suggest that FSHR expression levels in the blood vessels of CCRCC primary tumours can be used to predict, with high sensitivity and specificity, the patients who will respond to sunitinib therapy.

Analysis of pituitary gonadotropin concentration in blood serum and immunolocalization and immunoexpression of follicle stimulating hormone and luteinising hormone receptors in ovaries of postmenopausal women.

The participation of gonadotropins in ovarian carcinogenesis is well known and is supported by studies with inhibition of pituitary gonadotropin secretion, which results in a diminished risk of cancer. However, there are few data on localization and expression of Follicle Stimulating Hormone and Luteinising Hormone Receptors (FSHR and LHR) in ovaries of healthy postmenopausal women, and their correlation with FSH and LH concentration in blood serum is unknown. The aim of our study was to analyze gonadotropin concentration in blood serum and the expression of FSHR and LHR in ovaries of 207 postmenopausal women. Patients included in the study were divided into three groups depending on the number of years since menopause. We analyzed the concentration of FSH and LH in blood serum and the expression of FSHR and LHR in ovaries. Ovaries of postmenopausal women showed numerous morphological changes in the cortex and medulla when compared to the structure of ovaries of women at reproductive age. In all groups of patients clefts in the surface epithelium and epithelial inclusion cysts were found. The concentration of FSH and LH in the blood serum of women studied increased significantly with time from menopause. Significant differences between analyzed menopausal groups were found. The highest FSH and LH concentration in blood serum were found in women with the longest period of time from menopause. Quantitatively similar expression of FSHR and LHR was found in ovarian surface epithelial cells, in epithelial inclusion cysts and in the connective tissue cells of ovarian stroma. The intensity of the immunohistochemical reaction decreased with time from menopause and with age.