Sex hormone imbalances and adipose tissue dysfunction impacting on metabolic syndrome; a paradigm for the discovery of novel adipokines.

Sex hormone imbalance is causally related with visceral adipose tissue (AT) dysfunction and visceral obesity - an etiological component of metabolic syndrome (MetS), associated with high risk of both cardiovascular disease (CVD) and type 2 diabetes. In general, premenopausal women appear to be protected from CVD and the dramatic decline in sex steroid hormone occurring during menopausal transitions or other sex-related disorders influence the regional distribution, function, and metabolism of AT and increase the risk of CVD. Visceral AT dysfunction, manifesting as abnormality of fatty acid metabolism, increased oxidative stress, endothelial dysfunction, and excessive production of adipokines have been proposed in the pathogenesis of MetS. However, direct evidence of molecular mechanisms of depot-specific AT alterations, and dysfunction causally related to MetS is limited in studies on postmenopausal women due to difficulty in collecting discrete AT specimens at different ages and repeated sampling from different fat depots. This can be overcome using animal models that can mimic the cluster of pathology leading to MetS and help establish the molecular basis of links between loss of gonadal function on various AT depots and their contribution to MetS. Our group used sex hormone imbalance FSH receptor knock out (FORKO) female mice to recapitulate different aspects of the MetS and addressed the mechanism of visceral obesity related to MetS and discover two novel sex steroid hormone-regulated deep mesenteric estrogen-dependent adipose (MEDAs) genes. Taken together, such recent studies raise hopes for pharmacologic intervention strategies targeting sex steroid hormone signaling in AT to provide protection against AT dysfunction.

Bone marrow transplantation restores follicular maturation and steroid hormones production in a mouse model for primary ovarian failure.

Recent studies suggest that bone marrow stem cells (BMSCs) are promising grafts to treat a variety of diseases, including reproductive dysfunction. Primary ovarian failure is characterized by amenorrhea and infertility in a normal karyotype female, with an elevated serum level of follicle-stimulating hormone (FSH) and a decrease level of estrogen caused by a mutation in FSH receptor (FSHR) gene. Currently, there is no effective treatment for this condition. The phenotype of FSHR (-/-) mouse, FORKO (follitropin receptor knockout), is a suitable model to study ovarian failure in humans. Female FORKO mice have elevated FSH, decreased estrogen levels, are sterile because of the absence of folliculogenesis, and display thin uteri and small nonfunctional ovaries. In this study, we determined the effects of BMSC transplantation on reproductive physiology in this animal model. Twenty four hours post BMSC transplantation, treated animals showed detectable estroidogeneic changes in daily vaginal smear. Significant increase in total body weight and reproductive organs was observed in treated animals. Hemotoxylin and eosin (H&E) evaluation of the ovaries demonstrated significant increase in both the maturation and the total number of the follicles in treated animals. The FSH dropped to 40-50% and estrogen increased 4-5.5 times in the serum of treated animals compared to controls. The FSHR mRNA was detected in the ovaries of treated animals. Our results show that intravenously injected BMSCs were able to reach the ovaries of FORKO mice, differentiate and express FHSR gene, make FSHR responsive to FSH, resume estrogen hormone production, and restore folliculogenesis.

Initiation of the expression of peroxisome proliferator-activated receptor gamma (PPAR gamma) in the rat ovary and the role of FSH.

PPARgamma is highly expressed in granulosa cells by 23 days post-partum (pp) and is down-regulated in response to the LH surge. We tested the hypothesis that high levels of FSH during the neonatal period trigger the expression of PPARgamma. To determine when PPARgamma expression is initiated, ovaries were collected from neonatal rats. Messenger RNA for PPARgamma was undetectable on day 1, low from days 5-14, and increased by day 19 pp (p < 0.05). PPARgamma was detected in select granulosa cells in primary/early secondary follicles. Messenger RNA for the FSH receptor was detected as early as day 1 and remained steady throughout day 19 pp. The FSH receptor was detected by immunoblot analysis in ovaries collected 1, 2, and 5-9 days pp. In a subsequent experiment, neonatal rats were treated with acyline (GnRH antagonist) which significantly reduced FSH (p < 0.05) but not levels of mRNA for PPARgamma. The role of FSH in the induction of PPARgamma expression was further assessed in ovarian tissue from FORKO mice. Both mRNA and protein for PPARgamma were identified in ovarian tissue from FORKO mice. In summary, the FSH/FSH receptor system is present in granulosa cells prior to the onset of expression of PPARgamma. Reducing FSH during the neonatal period, or the ability to respond to FSH, did not decrease expression of mRNA for PPARgamma. These data indicate that FSH is not a primary factor initiating the expression of PPARgamma and that other agents play a role in activating its expression in the ovary.

Potentiation of vascular oxidative stress and nitric oxide-mediated endothelial dysfunction by high-fat diet in a mouse model of estrogen deficiency and hyperandrogenemia.

Estrogen deficiency is associated with increased cardiovascular risk due, in part, to hypertension, endothelial dysfunction, obesity, and hypercholesterolemia. Underlying mechanisms for this remain unclear. Here, we investigated whether high-fat intake aggravates vascular dysfunction through oxidative stress and inflammation, which could predispose to cardiovascular injury in conditions of estrogen deficiency, such as menopause. We studied female homozygous follitropin receptor knock out (FORKO) mice, which have hormonal features of clinical menopause and hypertension and wild-type (WT) and heterozygote mice (HTZ), fed a standard or high-fat diet for 4 months. Vascular function and structure were evaluated in arterial segments by pressurized myography. Acetylcholine (ACh)-induced vasodilation was reduced in FORKO vs. WT mice (P < .001). N(varpi)-nitro-l-arginine-methyl-ester inhibited Ach-induced relaxation in all groups on normal chow and in WT and HTZ on high-fat diet (FD) but had no effect in fat-fed FORKO mice. Antioxidant cocktail (superoxide dismutase, catalase, Tempol) increased ACh responses only in high-fat diet FORKO mice (P < .05). Vascular media-to-lumen ratio was increased and reactive oxygen species (ROS) generation, nitrotyrosine formation, and plasma nitrite levels were augmented in fat-fed FORKO vs. FORKO on normal chow. High-fat diet did not influence vascular inflammatory responses in any group. Our data demonstrate that FORKO mice have altered nitric oxide-sensitive vasorelaxation and vascular remodeling, which are aggravated by high-fat diet. Underlying mechanisms for this may involve decreased NO formation and increased generation of ROS and nitrotyrosine. These findings suggest that high-fat intake potentiates vascular damage in estrogen-deficient states, an effect involving increased oxidative stress.

Cardiac hypertrophy is associated with altered thioredoxin and ASK-1 signaling in a mouse model of menopause.

Oxidative stress is implicated in menopause-associated hypertension and cardiovascular disease. The role of antioxidants in this process is unclear. We questioned whether the downregulation of thioredoxin (TRX) is associated with oxidative stress and the development of hypertension and target-organ damage (cardiac hypertrophy) in a menopause model. TRX is an endogenous antioxidant that also interacts with signaling molecules, such as apoptosis signal-regulated kinase 1 (ASK-1), independently of its antioxidant function. Aged female wild-type (WT) and follitropin receptor knockout (FORKO) mice (20-24 wk), with hormonal imbalances, were studied. Mice were infused with ANG II (400 ng x kg(-1) x min(-1); 14 days). Systolic blood pressure was increased by ANG II in WT (166+/-8 vs. 121+/-5 mmHg) and FORKO (176+/-7 vs. 115+/-5 mmHg; P<0.0001; n=9/group) mice. In ANG II-infused FORKO mice, cardiac mass was increased by 42% (P<0.001). This was associated with increased collagen content and augmented ERK1/2 phosphorylation (2-fold). Cardiac TRX expression and activity were decreased by ANG II in FORKO but not in WT (P<0.01) mice. ASK-1 expression, cleaved caspase III content, and Bax/Bcl-2 content were increased in ANG II-infused FORKO (P<0.05). ANG II had no effect on cardiac NAD(P)H oxidase activity or on O(2)(*-) levels in WT or FORKO. Cardiac ANG II type 1 receptor expression was similar in FORKO and WT. These findings indicate that in female FORKO, ANG II-induced cardiac hypertrophy and fibrosis are associated with the TRX downregulation and upregulation of ASK-1/caspase signaling. Our data suggest that in a model of menopause, protective actions of TRX may be blunted, which could contribute to cardiac remodeling independently of oxidative stress and hypertension.

Impairment of the natriuretic peptide system in follitropin receptor knockout mice and reversal by estradiol: implications for obesity-associated hypertension in menopause.

Estrogen is considered a major regulator of adipose tissue in females. Estrogen increases circulating levels of atrial natriuretic peptide (ANP), a hormone with renal and cardiovascular effects. The aim of this study was to determine the status of the natriuretic peptide system in female follitropin-receptor knockout (FORKO) mice that could be associated with obesity and hypertension observed in these mutants. Furthermore, estradiol treatment was used to reverse alterations observed. FORKO and wild-type (WT) mice received daily injections of estradiol for 4 d. On the fifth day, blood was collected for determination of plasma ANP levels, and selected tissues were collected for determination of ANP, natriuretic peptide receptor type-A (NPR-A) and type-C (NPR-C) gene expression by RT-PCR and binding of [(125)I]ANP by autoradiography. At 5 months of age, FORKO mice were heavier and had more adipose tissue than WT mice. FORKO mice had lower plasma ANP levels and atrial ANP gene expression and higher renal and adipocyte NPR-C gene expression than WT mice. Estradiol treatment reduced weight gain and increased atrial ANP synthesis as well as decreased ANP clearance NPR-C receptors, resulting in elevation of circulating ANP level. In conclusion, this study shows that FORKO females have an impaired natriuretic peptide system, which may contribute to the susceptibility of FORKO mice to developing age-related hypertension previously shown in these animals. This study establishes a relation between estrogen, adipose tissue, and ANP, which may have important implications in menopausal women.

Changes in adiponectin and inflammatory genes in response to hormonal imbalances in female mice and exacerbation of depot selective visceral adiposity by high-fat diet: implications for insulin resistance.

Early obesity and late onset of insulin resistance associated with hormonal imbalances occur in FSH receptor-deficient follitropin receptor knockout female mice. This study tests the hypothesis that chronic high-fat diet aggravates obesogenic changes in a depot-specific manner and explores some molecular links of hormone imbalances with insulin resistance. In SV 129 mice, hormonal imbalances seem obligatory for exacerbation of diet-induced obesity. Visceral adiposity, glucose intolerance, and lipid disturbances in 9-month follitropin receptor knockout females were associated with decrease in adiponectin signaling. High-molecular-weight plasma adiponectin and adipose tissue adiponectin mRNA were decreased. Adiponectin receptors R1 and R2 mRNA was selectively altered in mesenteric fat but not periuterine fat. R2 decreased in the liver and R1 was higher in muscle. Whereas hepatic adenosine monophosphate T-activated protein kinase activity was down-regulated, both phosphoenolpyruvate carboxykinase and glucose-6-phosphatase enzymes were up-regulated. Longitudinally, diminishing sex hormone signaling in adipose tissue was associated with progressive down-regulation of adiponectin activity and gradual impaired glucose tolerance. Chronic high-fat diet in SV129 wild-type mice did not produce overt obesity but induced visceral fat depot changes accompanied by liver lipid accumulation, high cholesterol, and up-regulation of inflammation gene mRNAs. Thus, TNF-alpha, C-C motif chemokine receptor-2, and C-C motif chemokine ligand-2 were selectively elevated in mesenteric fat without altering glucose tolerance and adiponectin signaling. Our study highlights adiponectin signaling and regulation to be involved in hormone imbalance-induced insulin resistance and demonstrates selective visceral adipose depot alterations by chronic high-fat diet and induction of inflammatory genes.

Early alterations in ovarian surface epithelial cells and induction of ovarian epithelial tumors triggered by loss of FSH receptor.

Little is known about the behavior of the ovarian surface epithelium (OSE), which plays a central role in ovarian cancer etiology. It has been suggested that incessant ovulation causes OSE changes leading to transformation and that high gonadotropin levels during postmenopause activate OSE receptors, inducing proliferation. We examined the chronology of OSE changes, including tumor appearance, in a mouse model where ovulation never occurs due to deletion of follitropin receptor. Changes in epithelial cells were marked by pan-cytokeratin (CK) staining. Histologic changes and CK staining in the OSE increased from postnatal day 2. CK staining was observed inside the ovary by 24 days and increased thereafter in tumor-bearing animals. Ovaries from a third of aged (1 year) mutant mice showed CK deep inside, indicating cell migration. These tumors resembled serous papillary adenoma of human ovaries. Weak expression of GATA-4 and elevation of PCNA, cyclooxygenase-1, cyclooxygenase-2, and platelet-derived growth factor receptors alpha and beta in mutants indicated differences in cell proliferation, differentiation, and inflammation. Thus, we report that OSE changes occur long before epithelial tumors appear in FORKO mice. Our results suggest that neither incessant ovulation nor follicle-stimulating hormone receptor presence in the OSE is required for inducing ovarian tumors; thus, other mechanisms must contribute to ovarian tumorigenesis.

Impairments of heat shock protein expression and MAPK translocation in the central nervous system of follitropin receptor knockout mice.

The central nervous system is exposed to the chronic oxidative stress during aging when the endogenous defence weakens and the load of reactive oxygen species enhances. Sex hormones and heat shock proteins (Hsps) participate in these responses to stress. Their regulation is disturbed in aging. We assessed the expression of Hsps in hippocampus and cortex of follitropin receptor knockout (FORKO) mice, known to exhibit gender and age-dependent imbalance in sex steroids and gonadotropins. These imbalances could contribute to an impaired regulation of Hsps thereby increasing the risk of developing neurodegenerative disorders. Our study shows that, in the hippocampus the expression of Hsp70 and Hsp25 was reduced in 20-month-old FORKO mice. However, in the cortex both Hsps were significantly down regulated only in elderly females. There is a well-established co-regulation between Hsps and mitogen-activated protein kinases (MAPKs). Significant, gender-specific impairments in the translocation of phosphorylated ERK and JNK were found in the CNS structures in aged FORKO mice. Our results suggest that hormonal imbalances lead to a disturbed subcellular distribution of activated MAPKs which contribute to the impairments of signal transduction networks maintaining normal physiological functions in the cortex and hippocampus that are associated with neurodegenerative changes in aging.

Normalization of hormonal imbalances, ovarian follicular dynamics and metabolic effects in follitrophin receptor knockout mice.

Genetically modified follitrophin receptor knockout female mice with total FSH-receptor (FSH-R) deletion are sterile and their combined estrogen deficiency-hyperandrogenemic status provides an experimental paradigm to study the effect of hormonal imbalances on ovarian function and metabolic alterations. Elevated LH levels causing hyperandrogenemia perturb normal folliculogenesis. To control diverse pathophysiology associated with hormonal imbalances, we investigated the effects of transplanting a single normal mouse ovary in young mutants. An intact FSH-R signalling system in the graft responded promptly to the up-regulated pituitary gonadotrophins circulating in the host mutant. Resumption of regular estrous cycles validated stimulation of uterine functions. Secretions from the viable functioning grafts partially corrected follicular abnormalities originally present in host ovaries. Stromal hyperplasia responsible for high ovarian LH-receptor and key enzymes in host thecal/interstitial complex and hyperandrogenemia was reduced in host ovaries. Increases in plasma estradiol and reduced LH and free testosterone re-established the negative-feedback system. Reduced android obesity and activation of mammary glands indicated the combined beneficial effects of normalized steroid hormones on target organs. These data provide evidence that ovarian transplantation in mutants corrects estrogen loss and hyperandrogenemia. However, correction of hormonal imbalances is not sufficient to fully restore effects of FSH-R loss in host granulosa cells.

Altered ovarian function affects skeletal homeostasis independent of the action of follicle-stimulating hormone.

Osteoporosis is a leading public health problem. Although a major cause in women is thought to be a decline in estrogen, it has recently been proposed that FSH or follitropin is required for osteoporotic bone loss. We examined the FSH receptor null mouse (FORKO mouse) to determine whether altered ovarian function could induce bone loss independent of FSH action. By 3 months of age, FORKO mice developed age-dependent declines in bone mineral density and trabecular bone volume of the lumbar spine and femur, which could be partly reversed by ovarian transplantation. Bilateral ovariectomy reduced elevated circulating testosterone levels in FORKO mice and decreased bone mass to levels indistinguishable from those in ovariectomized wild-type controls. Androgen receptor blockade and especially aromatase inhibition each produced bone volume reductions in the FORKO mouse. The results indicate that ovarian secretory products, notably estrogen, and peripheral conversion of ovarian androgen to estrogen can alter bone homeostasis independent of any bone resorptive action of FSH.

The tale of follitropin receptor diversity: a recipe for fine tuning gonadal responses?

The original concept (dogma) of a single FSH receptor entity coupling to G(s) protein to activate adenylate cyclase and producing cAMP as second messenger appears inadequate to explain pleiotropic actions of the hormone. The identification and expression of alternatively spliced gonadotropin receptors, suggest that alternative splicing could serve as a mechanism for creating receptor diversity. Studies focused on sheep and mouse gonadal tissues show that the single large gene of approximately 250kb is a modular structure whose pre-mRNA undergoes alternative splicing creating several subtypes (at least four FSH-R1 to R4 identified to date). With segments of the N-terminus that are identical different topographies are generated by differing carboxyl termini. The same gene thus produces receptor types with different motifs that can display dominant positive, dominant negative, growth factor/cytokine type and potentially soluble binding protein features. Functional relevance is shown by modulation of receptor variants during hormonal stimulation. Presence of equivalent segments of the gene in the human and bovine suggests conservation and predicts similarity in structures and function. Thus, the complex cellular biology of follitropin receptors that may interact differently with polymorphic forms (glycosylation variants) of FSH represents an intricate scheme to regulate hormone signaling.

Increased blood pressure, vascular inflammation, and endothelial dysfunction in androgen-deficient follitropin receptor knockout male mice.

The relationship between testosterone, vascular function, and blood pressure remains unclear. Here we utilized a mouse model of andropause, follitropin receptor knockout (FORKO) male mice, which are testosterone-deficient, to investigate whether vascular function and structure are altered and whether this is associated with elevated blood pressure. Blood pressure was measured by radiotelemetry, and vascular function and structure were assessed in isolated pressurized mesenteric resistance arteries in wild-type (WT) and FORKO mice. Diastolic and mean arterial pressures were significantly higher in FORKO than in WT mice (P < .05). Resistance arteries of FORKO mice had greater media-to-lumen ratio (10.4 vs. 8.2; P < .05) and reduced relaxation responses to acetylcholine (Ach) (62% vs. 94% at Ach 10(-4) mol/L, P < .05) in pressurized preparations. N(omega)-nitro-L-arginine (L-NAME) reduced Ach-induced relaxation equally in both groups (45% to 46%), and plasma nitrite was lower (P < .05) in FORKO mice. However, the L-NAME-resistant relaxation was smaller in FORKO (16% vs. 48% at Ach 10(-4) mol/L, P < .05). In FORKO, expression of intracellular adhesion molecule-1 and vascular cell adhesion molecule-1 was enhanced by immunohistochemistry, and vascular estrogen receptors (ER)alpha/ERbeta expression ratio was decreased 5-fold by immunoblot analysis. Vasoconstrictor responses to angiotensin II were blunted, and angiotensin receptor 1 expression was decreased in FORKO mice. Our data indicate that in androgen-deficient FORKO mice, blood pressure is elevated and resistance arteries exhibit endothelial dysfunction, structural remodeling, and vascular inflammation. These phenomena may be related to reduced expression of ERalpha and/or to decreased testosterone levels and indicate that androgens may play an important role in modulating vascular function and regulation of blood pressure.

Early obesity and age-related mimicry of metabolic syndrome in female mice with sex hormonal imbalances.

OBJECTIVE: To investigate the relationship of early obesity to metabolic syndrome during sex hormonal imbalances in mutant female mice at different ages. RESEARCH METHODS AND PROCEDURES: Hormonal imbalances, accumulation and nature of adipose tissue, food intake, glucose tolerance, and expression of candidate genes and markers of inflammation were studied by comparing wild-type, null, and haploinsufficient follitropin receptor knockout female mice at different ages. RESULTS: Follitropin receptor deletion in mice produced null females that are infertile and haploinsufficient mice that undergo accelerated biological aging. Both types of mutants with sex hormonal imbalances have central obesity without hyperphagia, but circulating leptin is elevated. Adipocyte hyperplasia and hypertrophy is attributed to elevated peroxisome proliferator-activated receptor gamma expression. Adiponectin protein levels increase in fat tissue and plasma. Only mutants but not controls acquire age-dependent decline in glucose tolerance with high insulin and altered pancreatic beta cells. Changes in inflammation markers, decreased muscle insulin receptor phosphorylation, and increase of the enzyme protein tyrosine phosphatase 1B indicate insulin resistance. DISCUSSION: In this animal model, the chronological appearance of early obesity induced by hormonal imbalances culminates in characteristics that are attributable to metabolic syndrome, including cardiovascular abnormalities. Dissection of the depot-specific alterations and defining molecular interrelationships could help in developing targeted remedies and resolving complications and controversies related to health benefits and adversities of current hormone replacement therapy.

Angiotensin II induces vascular dysfunction without exacerbating blood pressure elevation in a mouse model of menopause-associated hypertension.

BACKGROUND: Follitropin-receptor knockout (FORKO) mice are estrogen-deficient, hyperandrogenic and exhibit features of menopause and elevated blood pressure (BP). Because the renin-angiotensin system has been implicated in menopause-associated hypertension, we questioned whether angiotensin II (Ang II) challenge would further increase BP in FORKO mice and whether this is associated with cardiovascular remodeling and inflammation. RESULTS: Ang II (400 ng/kg per min) increased BP, assessed by radiotelemetry, similarly in female FORKO and wild-type (WT) mice. Acetylcholine-induced vasodilation was attenuated and Ang II-induced contraction was enhanced in FORKO mice (P < 0.05). This was associated with increased expression of vascular Ang type 1 receptors (AT1R) and estrogen receptor alpha (ERalpha). Vascular structure (media/lumen ratio) was similar in both groups. Abundance of gp91, nitrotyrosine formation and superoxide production, indices of inflammation and cardiac collagen content were increased in Ang II-treated FORKO compared to Ang II-treated WT mice (P < 0.05). CONCLUSIONS: Thus, in FORKO mice Ang II exacerbates endothelial dysfunction, augments contractility, increases oxidative stress, and promotes cardiac fibrosis without worsening vascular remodeling or BP elevation compared to Ang II-treated WT controls. Our findings suggest that in FORKO mice Ang II may be more important in influencing vascular tone and endothelial function, possibly through oxidative stress and altered ERalpha signaling, than in arterial remodeling and BP elevation.

FSH directly regulates bone mass.

Postmenopausal osteoporosis, a global public health problem, has for decades been attributed solely to declining estrogen levels. Although FSH levels rise sharply in parallel, a direct effect of FSH on the skeleton has never been explored. We show that FSH is required for hypogonadal bone loss. Neither FSHbeta nor FSH receptor (FSHR) null mice have bone loss despite severe hypogonadism. Bone mass is increased and osteoclastic resorption is decreased in haploinsufficient FSHbeta+/- mice with normal ovarian function, suggesting that the skeletal action of FSH is estrogen independent. Osteoclasts and their precursors possess G(i2alpha)-coupled FSHRs that activate MEK/Erk, NF-kappaB, and Akt to result in enhanced osteoclast formation and function. We suggest that high circulating FSH causes hypogonadal bone loss.

Aberrant expression of PDGF ligands and receptors in the tumor prone ovary of follitropin receptor knockout (FORKO) mouse.

Although PDGF family members play a vital role in cell proliferation, motility and chemotaxis via activation of structurally similar alpha- and beta-receptors, little is known of their function in ovarian regulation and induction of tumorigenesis. Microarray analyses of ovaries from young follitropin receptor knockout (FORKO) mice that are prone to late ovarian tumors upon aging have revealed significant imbalances in PDGF ligands and receptors. We hypothesized that FSH/FSH-R signaling may exert effects partly by regulation of PDGF the family. To further understand their implications for ovarian tumorigenesis, we studied FORKO ovaries and hormonal regulation of the PDGF family members in normal mice, by using RT-PCR, Q-PCR, immunohistochemistry and western blotting. While PDGF-C and PDGFR-alpha increased, PDGFR-beta mRNA and protein decreased significantly in absence of FSH-R signaling. In the normal ovary, PDGFR-alpha was not affected by gonadotropin (eCG) stimulation but PDGF-C and PDGFR-beta decreased. Administration of estradiol decreased PDGF and their receptors. To further probe the differential regulation of PDGF family members by eCG and estradiol, we co-administered eCG with estrogen antagonist, ICI 182780. Increase in PDGFR-alpha in the absence of estradiol suggests direct effects of FSH signaling. During the estrous cycle in mice PDGF-C, PDGF-D and PDGFR-alpha mRNA levels were higher at the proestrous. By IHC, we report for the first time the localization of PDGF-C, PDGFR-alpha and PDGFR-beta protein in mouse ovarian compartments including the surface epithelium that is also altered in mutants. Immunostaining of PDGFRs increased as the follicle developed to preantral stage and declined thereafter. Thus, FSH modulates PDGF family members, partly via E2, suggesting that loss of FSH-R signaling causes an imbalance of PDGF family members predisposing the abnormal ovarian follicular environment for inducing tumorigenesis in aging FORKO mice.

Differential expression of claudin family proteins in mouse ovarian serous papillary epithelial adenoma in aging FSH receptor-deficient mutants.

Ovarian cancer is a deadly disease with long latency. To understand the consequences of loss of follicle-stimulating hormone receptor (FSH-R) signaling and to explore why the atrophic and anovulatory ovaries of follitropin receptor knockout (FORKO) mice develop different types of ovarian tumors, including serous papillary epithelial adenoma later in life, we used mRNA expression profiling to gain a comprehensive view of misregulated genes. Using real-time quantitative reverse transcription-polymerase chain reaction, protein analysis, and cellular localization, we show, for the first time, in vivo evidence that, in the absence of FSH-R signaling, claudin-3, claudin-4, and claudin-11 are selectively upregulated, whereas claudin-1 decreases in ovarian surface epithelium and tumors in comparison to wild type. In vitro experiments using a mouse ovarian surface epithelial cell line derived from wild-type females reveal direct hormonal influence on claudin proteins. Although recent studies suggest that cell junction proteins are differentially expressed in ovarian tumors in women, the etiology of such changes remains unclear. Our results suggest an altered hormonal environment resulting from FSH-R loss as a cause of early changes in tight junction proteins that predispose the ovary to late-onset tumors that occur with aging. More importantly, this study identifies claudin-11 overexpression in mouse ovarian serous cystadenoma.

Targeting gonadotropin receptor genes: reproductive biology, aging, and related health implications.

This review highlights observations gleaned from recent reports on the deletion of FSH and LH receptors in mice. Gonadal differentiation does not depend on the presence of gonadotropin receptors but development is affected to varying degrees in both sexes. In both knockouts the null females are infertile with severely underdeveloped gonads and accessory structures. Sexual maturity and/or pubertal delay occur depending on the sex and knockout. Male null FSH-R mice have reduced fertility but null LH-R males are sterile due to cryptorchid testes and deficient spermatogenesis. In null FSH-R females hormonal imbalances are due to deficient estrogen and hyperandrogenemia. LH-R deficient females have low estrogen and testosterone. Females in both knockouts display phenotypes such as obesity, bone deficiency, and changes in brain structure and function in addition to manifestation of different types of reproductive tract tumors. Both types of mice represent good models for testing hormone replacement therapy in different combinations. The FSH-R heterozygous females could also be useful for studying age-dependent phenotypes.

Effects of FSH receptor deletion on epididymal tubules and sperm morphology, numbers, and motility.

Follicle stimulating hormone (FSH) interacts with its cognate receptor (R) on Sertoli cells within the testis and plays an important role in the maintenance of spermatogenesis. Male FSH-R knockout (FORKO) mice show fewer Sertoli cells and many that are structurally abnormal and as a consequence fewer germ cells. Lower levels of serum testosterone (T) and androgen binding protein (ABP) also occur, along with reduced fertility. To assess the effects of FSH-R depletion as an outcome of testicular abnormalities, sperm from the cauda epididymidis were counted and examined ultrastructurally. As reduced fertility may also reflect changes to the epididymis, the secondary responses of the epididymis to lower T and ABP levels were also examined by comparing differences in sizes of epididymal tubules in various regions of FORKO and wild type (WT) mice. Sperm motility was evaluated in FORKO mice and compared to that of WT mice by computer assisted sperm analysis (CASA). Quantitatively, the data revealed that epithelial areas of the caput and corpus epididymidis were significantly smaller in FORKO mice compared to WT mice. Cauda epididymal sperm counts in FORKO mice were also much lower than in WT mice. This resulted in changes to 9 out of 14 sperm motility parameters, related mostly to velocity measures, which were significantly lower in the FORKO mice. The greatest change was observed relative to the percent static sperm, which was elevated by 20% in FORKO mice compared to controls. EM analyses revealed major changes to the structure of the heads and tails of cauda luminal sperm in FORKO mice. Taken together these data suggest a key role for the FSH receptor in maintaining Sertoli cells to sustain normal sperm numbers and proper shapes of their heads and tails. In addition, the shrinkage in epididymal epithelial areas observed in FORKO mice likely reflect direct and/or indirect changes in the functions of these cells and their role in promoting sperm motility, which is noticeably altered in FORKO mice.