Renato M. Salgado

Modulation of small leucine-rich proteoglycans (SLRPs) expression in the mouse uterus by estradiol and progesterone

BackgroundWe have previously demonstrated that four members of the family of small leucine-rich-proteoglycans (SLRPs) of the extracellular matrix (ECM), named decorin, biglycan, lumican and fibromodulin, are deeply remodeled in mouse uterine tissues along the estrous cycle and early pregnancy. It is known that the combined action of estrogen (E2) and progesterone (P4) orchestrates the estrous cycle and prepares the endometrium for pregnancy, modulating synthesis, deposition and degradation of various molecules. Indeed, we showed that versican, another proteoglycan of the ECM, is under hormonal control in the uterine tissues.MethodsE2 and/or medroxiprogesterone acetate (MPA) were used to demonstrate, by real time PCR and immunoperoxidase staining, respectively, their effects on mRNA expression and protein deposition of these SLRPs, in the uterine tissues.ResultsDecorin and lumican were constitutively expressed and deposited in the ECM in the absence of the ovarian hormones, whereas deposition of biglycan and fibromodulin were abolished from the uterine ECM in the non-treated group. Interestingly, ovariectomy promoted an increase in decorin, lumican and fibromodulin mRNA levels, while biglycan mRNA conspicuously decreased. Hormone replacement with E2 and/or MPA differentially modulates their expression and deposition.ConclusionsThe patterns of expression of these SLRPs in the uterine tissues were found to be hormone-dependent and uterine compartment-related. These results reinforce the existence of subpopulations of endometrial fibroblasts, localized into distinct functional uterine compartments, resembling the organization into basal and functional layers of the human endometrium.

The Estrous Cycle Modulates Small Leucine‐Rich Proteoglycans Expression in Mouse Uterine Tissues

In the pregnant mouse uterus, small leucine‐rich proteoglycans (SLRPs) are drastically remodeled within a few hours after fertilization, suggesting that ovarian hormone levels modulate their synthesis and degradation. In this study, we followed by immunoperoxidase approach, the presence of four members of the SLRP family (decorin, lumican, biglycan, and fibromodulin) in the uterine tissues along the estrous cycle of the mouse. All molecules except fibromodulin, which predominates in the myometrium, showed a striking modulation in their distribution in the endometrial stroma, following the rise in the level of estrogen. Moreover, notable differences in the distribution of SLRPs were observed between superficial and deep stroma, as well as between the internal and external layers of the myometrium. Only biglycan and fibromodulin were expressed in the luminal and glandular epithelia. All four SLRPs were found in cytoplasmic granules of mononucleated cells. The pattern of distribution of the immunoreaction for these molecules in the uterine tissues was found to be estrous cycle‐stage dependent, suggesting that these molecules undergo ovarian hormonal control and probably participate in the preparation of the uterus for decidualization and embryo implantation. In addition, this and previous results from our laboratory suggest the existence of two subpopulations of endometrial fibroblasts that may be related to the centrifugal development of the decidua. Anat Rec, 2008.

Hormone-regulated expression and distribution of versican in mouse uterine tissues

BackgroundRemodeling of the extracellular matrix is one of the most striking features observed in the uterus during the estrous cycle and after hormone replacement. Versican (VER) is a hyaluronan-binding proteoglycan that undergoes RNA alternative splicing, generating four distinct isoforms. This study analyzed the synthesis and distribution of VER in mouse uterine tissues during the estrous cycle, in ovariectomized (OVX) animals and after 17beta-estradiol (E2) and medroxyprogesterone (MPA) treatments, either alone or in combination.MethodsUteri from mice in all phases of the estrous cycle, and animals subjected to ovariectomy and hormone replacement were collected for immunoperoxidase staining for versican, as well as PCR and quantitative Real Time PCR.ResultsIn diestrus and proestrus, VER was exclusively expressed in the endometrial stroma. In estrus and metaestrus, VER was present in both endometrial stroma and myometrium. In OVX mice, VER immunoreaction was abolished in all uterine tissues. VER expression was restored by E2, MPA and E2+MPA treatments. Real Time PCR analysis showed that VER expression increases considerably in the MPA-treated group. Analysis of mRNA identified isoforms V0, V1 and V3 in the mouse uterus.ConclusionThese results show that the expression of versican in uterine tissues is modulated by ovarian steroid hormones, in a tissue-specific manner. VER is induced in the myometrium exclusively by E2, whereas MPA induces VER deposition only in the endometrial stroma.

Triiodothyronine Rapidly Alters the TSH Content and the Secretory Granules Distribution in Male Rat Thyrotrophs by a Cytoskeleton Rearrangement-Independent Mechanism

Rapid actions of T3 on TSH synthesis in posttranscriptional steps, such as polyadenylation and translation rate, have already been described. The focus of this paper was to characterize rapid actions of T3 on TSH secretion and the involvement of actin and microtubule cytoskeleton in this process. For that, sham-operated (SO) and thyroidectomized (Tx) rats were subjected to acute or chronic treatment with T3. We observed a disarrangement in microtubule and actin cytoskeletons and an increase in Tshb mRNA levels in Tx rats, whereas the total TSH protein content was reduced in the pituitary gland as a whole, but increased in the secretory granules close to the plasma membrane of thyrotrophs, as well as in the extracellular space. The acute T3 dose promoted a rapid increase and redistribution of TSH secretory granules throughout the cytoplasm, as well as a rearrangement in actin and microtubule cytoskeletons. The T3 chronic treatment outcome reinforces the acute effects observed and, additionally, evinces an increase in the α-tubulin content and a rearrangement in microtubule cytoskeleton. Thus, T3 is able to rapidly suppress TSH secretion and, in parallel, to promote a rearrangement in actin and microtubules assembly throughout the pituitary gland, effects that seem to be independent from each other.

Long-term type 1 diabetes impairs decidualization and extracellular matrix remodeling during early embryonic development in mice.

INTRODUCTION Endometrial decidualization and associated extracellular matrix (ECM) remodeling are critical events to the establishment of the maternal-fetal interface and successful pregnancy. Here, we investigated the impact of type 1 diabetes on these processes during early embryonic development, in order to contribute to the understanding of the maternal factors associated to diabetic embryopathies. METHODS Alloxan-induced diabetic Swiss female mice were bred after different periods of time to determine the effects of diabetes progression on the development of gestational complications. Furthermore, the analyses focused on decidual development as well as mRNA expression, protein deposition and ultrastructural organization of decidual ECM. RESULTS Decreased number of implantation sites and decidual dimensions were observed in the group mated 90-110 days after diabetes induction (D), but not in the 50-70D group. Picrosirius staining showed augmentation in the fibrillar collagen network in the 90-110D group and, following immunohistochemical examination, that this was associated with increase in types I and V collagens and decrease in type III collagen and collagen-associated proteoglycans biglycan and lumican. qPCR, however, demonstrated that only type I collagen mRNA levels were increased in the diabetic group. Alterations in the molecular ratio among distinct collagen types and proteoglycans were associated with abnormal collagen fibrillogenesis, analyzed by transmission electron microscopy. CONCLUSIONS Our results support the concept that the development of pregnancy complications is directly related with duration of diabetes (progression of the disease), and that this is a consequence of both systemic factors (i.e. disturbed maternal endocrine-metabolic profile) and uterine factors, including impaired decidualization and ECM remodeling.

Effects of long-term diabetes on the structure and cell proliferation of the myometrium in the early pregnancy of mice.

It is known that the development of diabetic complications in human pregnancy is directly related to the severity and the duration of this pathology. In this study, we developed a model of long‐term type 1 diabetes to investigate its effects on the cytoarchitecture, extracellular matrix and cell proliferation during the first adaptation phase of the myometrium for pregnancy. A single dose of alloxan was used to induce diabetes in mice prior to pregnancy. To identify the temporal effects of diabetes the mice were divided into two groups: Group D1 (females that became pregnant 90–100 days after alloxan); Group D2 (females that became pregnant 100–110 days after alloxan). Uterine samples were collected after 168 h of pregnancy and processed for light and electron microscopy. In both groups the histomorphometric evaluation showed that diabetes promoted narrowing of the myometrial muscle layers which was correlated with decreased cell proliferation demonstrated by PCNA immunodetection. In D1, diabetes increased the distance between muscle layers and promoted oedema. Contrarily, in D2 the distance between muscle layers decreased and, instead of oedema, there was a markedly deposition of collagen in the myometrium. Ultrastructural analysis showed that diabetes affects the organization of the smooth muscle cells and their myofilaments. Consistently, the immunoreaction for smooth muscle α‐actin revealed clear disorganization of the contractile apparatus in both diabetic groups. In conclusion, the present model demonstrated that long‐term diabetes promotes significant alterations in the myometrium in a time‐sensitive manner. Together, these alterations indicate that diabetes impairs the first phenotypic adaptation phase of the pregnant myometrium.

Long-term type 1 diabetes alters the deposition of collagens and proteoglycans in the early pregnant myometrium of mice.

INTRODUCTION We have previously shown that long-term type 1 diabetes affects the structural organization, contractile apparatus and extracellular matrix (ECM) of the myometrium during early pregnancy in mice. OBJECTIVE This study aimed to identify which myometrial ECM components are affected by diabetes, including fibril-forming collagen types I, III and V, as well as proteoglycans, decorin, lumican, fibromodulin and biglycan. METHODS Alloxan-induced type 1 diabetic female mice were divided into subgroups D1 and D2, formed by females that bred 90-100 and 100-110 days after diabetes induction, respectively. The deposition of ECM components in the myometrium was evaluated by immunohistochemistry/immunofluorescence. RESULTS The subgroup D1 showed decreased deposition of collagen types I and III in the external muscle layer (EML) and decreased collagen types III and V in the internal muscle layer (IML). Collagen types I and III were decreased in both muscle layers of the subgroup D2. In addition, increased deposition of collagen types I and III and lumican as well as decreased collagen type V were observed in the connective tissue between muscle layers of D2. Lumican was decreased in the EML of the subgroups D1 and D2. Fibromodulin was repressed in the IML and EML of both D1 and D2. In contrast, decorin deposition diminished only in muscle layers of D2. No changes were noticed for biglycan. CONCLUSIONS Subgroups D1 and D2 showed distinct stages of progression of diabetic complications in the myometrium, characterized by both common and specific sets of changes in the ECM composition.

Triiodothyronine differentially modulates the LH and FSH synthesis and secretion in male rats

Hypothyroidism and thyrotoxicosis produce adverse effects in male reproduction by unknown mechanisms. We investigated whether triiodothyronine (T3) modulates luteinizing hormone (LH) and follicle stimulating hormone (FSH) synthesis/secretion, by inducing different thyroid states. In hypothyroidism, the content of Lhb and Fshb mRNAs was increased, while their association to ribosomes and the protein content were reduced and the serum LH and FSH concentrations were augmented and decreased, respectively. Thyrotoxicosis reduced Lhb mRNA and LH serum concentration, and increased Lhb mRNA translational rate. The Fshb mRNA content and its association to ribosomes were also increased, whereas FSH serum concentrations were comparable to euthyroid levels. Acute T3 treatment decreased the total content of Lhb and Fshb mRNAs, and increased their association to ribosomes, as well as the LHB and FSHB contents in secretory granules. This study shows that T3 acts on gonadotrophs, resulting in direct effects on LH and FSH synthesis/secretion of male rats, suggesting that some reproductive disorders observed in men may be associated with thyroid hormone imbalances.