Cervical fluid pH, electrolytes and osmolarity changes and expression of ion transporters (ENaC, CFTR and AQP) in cervix of women with primary unexplained infertility.

BACKGROUND: Unexplained infertility could arise from a defect in the cervix. However, the contribution of abnormal cervical fluid microenvironment to this problem still needs to be identified. Therefore, this study identifies the changes in the cervical fluid microenvironment, i.e., pH, electrolytes and osmolarity as well as expression of ion transporters in the cervix including ENaC, CFTR and AQP in fertile women and in women suffering from primary unexplained infertility. METHODS: Fertile women and women with unexplained infertility but having regular 28-day menstrual cycles were chosen in this study, Day-22 serum progesterone levels were determined. In the meantime, serum FSH and LH levels were determined on day 2 while, cervical flushing was performed at day 14 to analyse changes in the cervical fluid pH, osmolarity, Na+ and Cl- levels. Meanwhile, cells retrieved from cervical fluid were subjected to mRNA expression and protein distribution analysis for CFTR, AQP and ENaC by qPCR and immunofluorescence, respectively. RESULTS: No significant changes in serum progesterone, FSH and LH levels were observed between the two groups. However, cervical fluid pH, osmolarity, Na+ and Cl- levels were significantly lower in primary unexplained infertile group when compared to fertile group. Expression of CFTR and AQP (AQP 1, AQP 2, AQP 5 and AQP 7) in endocervical cells was lower and expression of ß-ENaC was higher in primary unexplained infertile women (p < 0.05 when compared to fertile group). CONCLUSIONS: Alterations in the cervical fluid microenvironment linked to the defective ion transporter expression in the cervix might contribute towards the unfavourable condition that accounts for unexplained infertility in women.

Oral administration of Centella asiatica (L.) Urb leave aqueous extract ameliorates cerebral oxidative stress, inflammation, and apoptosis in male rats with type-2 diabetes.

Centella asiatica is claimed to have a neuroprotective effect; however, its ability to protect the cerebrum against damage in diabetes has never been identified. The aims were to identify the possibility that C. asiatica ameliorates inflammation, oxidative stress, and apoptosis in the cerebrum in diabetes. C. asiatica leave aqueous extract (C. asiatica) (50, 100, and 200 mg/kg/b.w.) were given to diabetic rats for 28 days. Changes in rats' body weight, food and water intakes, and insulin and FBG levels were monitored. Following sacrificed, cerebrum was harvested and subjected for histological, biochemical, and molecular biological analyses. The results revealed treatment with C. asiatica was able to ameliorate the loss in body weight, the increase in food and water intakes, the decrease in insulin, and the increase in FBG levels in diabetic rats. Additionally, histopathological changes in the cerebrum and levels of p38, ERK, JNK, cytosolic Nrf2, Keap-1, LPO, RAGE, and AGE levels decreased; however, PI3K, AKT, IR, IRS, GLUT-1, nuclear Nrf2, Nqo-1, Ho-1, and anti-oxidative enzymes (SOD, CAT, and GPx) levels increased in diabetic rats receiving C. asiatica. Furthermore, C. asiatica treatment also caused cerebral inflammation and apoptosis to decrease as indicated by decreased inflammatory markers (cytosolic NF-κB p65, p-Ikkß, Ikkß, iNOS, COX-2, TNF-α, IL-6, and IL-1ß), decreased pro-apoptosis markers (Casp-3, 9, and Bax), but increased anti-apoptosis marker, Bcl-2. Activity level of Na+/K+, Mg2+, and Ca2+-ATPases in the cerebrum also increased by C. asiatica treatment. Conclusions: C. asiatica treatment helps to prevent cerebral damage and maintain near normal cerebral function in diabetes.

Expression of proteins related to thyroid hormone function in the uterus is down-regulated at the day of implantation in hypothyroid pregnant rats.

Hypothyroidism has been linked to infertility, but the mechanisms underlying infertility-related hypothyroidism have yet to be fully elucidated. Therefore, in this study, effects of hypothyroidism on expression of the proteins related to thyroid hormone function in the uterus, which were thought to play a role implantation, including thyroid hormone receptor (TR), thyroid stimulating hormone receptor (TSHR), retinoic acid receptor (RAR) and extracellular kinase (ERK) were identified. Pregnant female rats were rendered hypothyroid by giving methimazole (MMI), orally. Following hypothyroid induction, rats were grouped into control (non-treated) and received subcutaneous thyroxine at 20, 40, and 80 µg/kg/day for five consecutive days. At Day 6, which is the day of implantation (GD 6), rats were sacrificed and the number of embryo implantation site in the uterus was calculated. Then, uterine horns were harvested and expression of the above proteins and their mRNAs were identified by Western blotting and real-time PCR, respectively. In non-treated hypothyroid pregnant rats, the number of embryo implantation sites decreased as compared to euthyroid and hypothyroid rats receiving thyroxine treatment. Similarly, expression of TRα-1, TRß-1, TSHR, ERK1/2 and RAR proteins and mRNA in the uterus of non-treated hypothyroid rats also decreased (P < 0.05 when compared to euthyroid and thyroxine-treated hypothyroid rats). In conclusion, downregulated expression of the thyroid hormone related proteins in the uterus at the day of implantation might result in infertility as reported in hypothyroid condition.

Effects of testosterone on mean arterial pressure and aquaporin (AQP)-1, 2, 3, 4, 6 and 7 expressions in the kidney of orchidectomized, adult male Sprague-Dawley rats.

We hypothesized that higher blood pressure in males than females could be due to testosterone effects on aquaporin (AQP) expression in kidneys. METHODS: Orchidectomized adult male Sprague-Dawley (SD) rats received seven days subcutaneous testosterone treatment (125 µg/kg/day or 250 µg/kg/day), with or without flutamide or finasteride. Following completion of treatment, MAP was determined in rats under anaesthesia via carotid artery cannulation. In another cohort of rats, kidneys were removed following sacrifice and AQP-1, 2, 3, 4, 6 and 7 protein and mRNA levels were determined by Western blotting and Real-time PCR respectively. Distribution of AQP subunits' protein in the nephrons were visualized by immunofluorescence. RESULTS: Testosterone caused MAP, AQP-1, 2, 4, 6 and 7 protein and mRNA levels in kidneys to increase while AQP-3 protein and mRNA levels in kidneys to decrease (p < 0.05). AQP-1 and 7 were found to be distributed in the proximal convoluted tubule (PCT) while AQP-2, 3, 4 and 6 were found to be distributed in the collecting ducts (CD). Effects of testosterone were antagonized by flutamide and finasteride. CONCLUSIONS: Elevated expression of AQP-1, 2, 4, 6 and 7 under testosterone influence in kidneys could lead to increase H2O reabsorption which eventually lead to increase in blood pressure.

Isoflavone genistein inhibits estrogen-induced chloride and bicarbonate secretory mechanisms in the uterus in rats.

We hypothesized that genistein could affect the chloride (Cl- ) and bicarbonate (HCO3- ) secretory mechanisms in uterus. Ovariectomized female rats were given estradiol or estradiol plus progesterone with 25, 50, or 100 mg/kg/day genistein. Following completion of the treatment, uterine fluid Cl- and HCO3- concentrations were determined by in vivo uterine perfusion. Uteri were subjected for molecular biological analysis (Western blot, qPCR, and immunohistochemistry) to detect levels of expression of Cystic Fibrosis transmembrane regulator (CFTR), Cl- /HCO3- exchanger (SLC26a6), Na+ /HCO3- cotransporter (SLC4a4), and estrogen receptor (ER)-α and ß. Coadministration of genistein resulted in decrease in Cl- and HCO3- concentrations and expression of CFTR, SLC26a6, SLC4a4, and ER-α and ER-ß in the uteri of estradiol-treated rats. In estradiol plus progesterone-treated rats, a significant increase in the above parameters were observed following high-dose genistein treatment except for the SLC24a4 level. In conclusion, genistein-induced changes in the uterus could affect the reproductive processes that might result in infertility.

Combinatorial effect of genistein and female sex-steroids on uterine fluid volume and secretion rate and aquaporin (AQP)-1, 2, 5, and 7 expression in the uterus in rats.

We hypothesized that genistein can interfere with the regulation of uterine fluid volume, secretion rate and expression of aquaporin in the uterus by female sex-steroids, i.e., estrogen and progesterone. Therefore, the aims of this study were to investigate changes in these parameters in the presence of genistein and female sex-steroids. METHODS: Female Sprague-Dawley rats were ovariectomized and received 3-days estradiol-17ß benzoate (E2) plus genistein (25, 50, or 100 mg kg-1  day-1 ) or 3-days E2 followed by 3-days E2 plus progesterone with genistein (25, 50, or 100 mg kg-1  day-1 ). A day after last treatment, uterine fluid secretion rate was determined by in vivo uterine perfusion with rats under anesthesia. Animals were sacrificed and uteri were harvested and subjected for histological analyses. Luminal/outer uterine circumference was determined and distribution of AQP-1, 2, 5, and 7 in endometrium was visualized by immunofluorescence. Expression of AQP-1, 2, 5, and 7 proteins and mRNAs were determined by Western blotting and Real-time PCR respectively. RESULTS: Combined treatment of E2 with high dose genistein (50 and 100 mg kg-1  day-1 ) resulted in significant decrease in uterine fluid volume, secretion rate and expression of AQP-1, 2, 5, and 7 proteins and mRNAs in uterus (p < 0.05). No significant changes in these parameters were observed when 25 mg kg-1  day-1 genistein was given with E2 or when genistein was given with E2 followed by E2 plus progesterone Conclusions: Decreased in uterine fluid volume, secretion rate and AQP-1, 2, 5, and 7 expression in the uterus by high dose genistein in the presence of E2 could potentially affect female fertility. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 832-844, 2017.

Vacuolar-ATPase (V-ATPase) Mediates Progesterone-Induced Uterine Fluid Acidification in Rats.

We hypothesized that progesterone-induced decrease in uterine fluid pH involves V-ATPase. In this study, expression and functional activity of V-ATPase in uterus were investigated under progesterone influence. Ovariectomized adult female rats received subcutaneous injection of estradiol-17ß (1 µg/kg/day) or progesterone (20 mg/kg/day) for 3 days or 3 days estradiol-17ß followed by 3 days vehicle, progesterone, or estradiol-17ß plus progesterone. Mifepristone, a progesterone receptor blocker, was concomitantly given to the rats which received progesterone. A day after last injection, rate of uterine fluid secretion, its HCO3 (-) concentration, and pH were determined via in vivo uterine perfusion in rats under anesthesia. V-ATPase inhibitor, bafilomycin, was introduced into the perfusion buffer, and changes in these parameters were observed. Expression of V-ATPase A1 and B1/2 proteins and mRNAs in uterus were quantified by Western blotting and real-time PCR, respectively. Distribution of these proteins was observed by immunohistochemistry. Our findings showed that under progesterone influence, uterine fluid secretion rate, HCO3 (-) concentration, and pH were significantly reduced. Administration of bafilomycin did not cause significant changes in fluid secretion rate; however, HCO3 (-) concentration and pH were significantly elevated. In parallel with these changes, expression of V-ATPase A1 and B1/2 proteins and mRNAs were significantly increased with these proteins highly distributed in uterine luminal and glandular epithelia. In conclusion, increased expression and functional activity of V-ATPase were most likely responsible for the decreased in uterine fluid pH observed under progesterone influence.

Testosterone Induces Increase in Aquaporin (AQP)-1, 5, and 7 Expressions in the Uteri of Ovariectomized Rats.

Testosterone has been reported to cause a decrease in uterine fluid volume in which this could involve the aquaporins (AQPs). This study aimed to investigate effect of testosterone on uterine AQP-1, 5, and 7 expressions in order to explain the reported reduction in uterine fluid volume under testosterone influence. Ovariectomized adult female rats received peanut oil, testosterone (1 mg/kg/day), estrogen (0.2 µg/kg/day), or combined estrogen plus testosterone for three consecutive days. Other groups received 3 days estrogen followed by 2 days either peanut oil or testosterone with or without flutamide or finasteride. A day after last injection, uteri were harvested, and the levels of AQP-1, 5, and 7 messenger RNA (mRNA) in uterine tissue homogenates were analyzed by real-time PCR (qPCR). Distributions of AQP-1, 5, and 7 proteins in uterus were observed by immunofluorescence. Levels of AQP-1 mRNA were elevated in rats receiving either estrogen or testosterone-only treatment; however, levels of AQP-5 and 7 mRNAs were elevated in rats receiving testosterone-only treatment. In rats pre-treated with estrogen, testosterone treatment resulted in higher AQP-1, 5, and 7 mRNA levels compared to vehicle treatment. Testosterone effects were antagonized by flutamide but not finasteride. Immunofluorescence study showed that AQP-1 was highly distributed in uterine lumenal epithelium following estrogen or testosterone-only treatment. However, AQP-5 and 7 distributions were high in uterine lumenal epithelium following testosterone-only treatment. Testosterone-induced up-regulation of AQP-1, 5, and 7 expressions in uterus could explain the observed reduction in uterine fluid volume as reported under this condition.

Estrogen and progesterone differentially regulate the levels of cystic fibrosis transmembrane regulator (CFTR), adenylate cyclase (AC), and cyclic adenosine mono-phosphate (cAMP) in the rat cervix.

The consistency of the cervical mucus changes with the reproductive cycle, which we hypothesized involved changing levels of cystic fibrosis transmembrane regulator (CFTR), adenylate cyclase (AC), and cyclic adenosine mono-phosphate (cAMP). We therefore measured the abundance of each in the rat cervix under estrogen and progesterone influence to determine if the activity of these components could explain the changes in the consistency of cervical mucus. Ovariectomised adult female rats were treated with three days of either estrogen (1 µg/kg/day) or progesterone (20 mg/kg/day), or three days of estrogen followed by two days of either vehicle or progesterone or estrogen plus progesterone. In some groups, mifepristone (7 mg/kg/day) was concurrently given with progesterone. Animals were then sacrificed, and the cervix was harvested for protein and mRNA expression analyses by Western blot and real-time PCR, respectively. The distribution of proteins was investigated by immunohistochemistry, and levels of cAMP were determined by enzyme-linked immunosorbent assay (ELISA). Cftr mRNA, AC protein, and cAMP levels in cervical homogenates as well as the tissue distribution of CFTR and AC in endocervical epithelia were highest under estrogen influence; the opposite pattern was seen under progesterone influence. Cervical lumen circumference was highest under estrogen and lowest under progesterone. The effects of progesterone were antagonized by mifepristone. Therefore, increased abundance of CFTR, AC, and cAMP under estrogen influence could account for the increased fluid accumulation within the cervical lumen, which would contribute to lower cervical mucus consistency, whereas progesterone reverses this effect at the molecular and organ level.

Bisphenol A, Dichlorodiphenyltrichloroethane (DDT) and Vinclozolin Affect ex-vivo Uterine Contraction in Rats via Uterotonin (Prostaglandin F2α, Acetylcholine and Oxytocin) Related Pathways.

UNLABELLED: Bisphenol-A (BPA), dichrolodiphenyltrichloroethane (DDT) and vinclozolin were found able to induce abnormal uterine contraction. The mechanisms involved remains unclear. We hypothesized that the effect of these compounds were mediated via the uterotonin pathways. Therefore, in this study, effects of BPA, vinclozolin and DDT-only and in combination with uterotonins (PGF-2α, acetylcholine and oxytocin) on the force and pattern of uterine contraction were observed. METHODS: Uteri were harvested from intact adult female rats 24 hours after a single injection (1 mg/kg/b.w) of estrogen to synchronize their oestrous cycle. The uterine horns were subjected for ex-vivo contraction studies in an organ bath connected to Powerlab data acquisition system. Different doses of BPA, vinclozolin and DDT were added into the bathing solution and changes in the pattern and strength of uterine contraction were recorded. Further, increasing doses of uterotonins were concomitantly administered with these compounds and changes in the force and pattern of contraction were observed. RESULTS: In the absence of uterotonins, uterine contractile force decreased with increasing doses of BPA and DDT. However, vinclozolin induced sharp increase in the contractile forces which then gradually decrease. Administration of BPA, DDT and vinclozolin alone reduced the force of uterine contraction following stimulation of contraction by uterotonins. However, BPA, vinclozolin or DDT effects were relieved upon co-administration with uterotonins at increasing doses. CONCLUSIONS: The antagonizing effect of uterotonins on BPA, vinclozolin and DDT actions could explain the mechanism underlying the adverse effect of these compounds on uterine contraction.

Enhanced Expression of Sodium Hydrogen Exchanger (NHE)-1, 2 and 4 in the Cervix of Ovariectomised Rats by Phytoestrogen Genistein.

UNLABELLED: Restoring the pH of cervicovaginal fluid is important for the cervicovaginal health after menopause. Genistein, which is a widely consumed dietary health supplement to overcome the post-menopausal complications could help to restore the cervicovaginal fluid pH. We hypothesized that genistien effect involves changes in expression of NHE-1, 2 and 4 proteins and mRNAs in the cervix. This study investigated effect of genistein on NHE-1, 2 and 4 protein and mRNA expression in the cervix in order to elucidate the mechanisms underlying possible effect of this compound on cervicovaginal fluid pH after menopause. METHODS: Ovariectomised adult female rats received 25, 50 and 100 mg/kg/day genistein for seven consecutive days. At the end of the treatment, animals were sacrificed and cervix was harvested. Expression of Nhe-1, 2 and 4 mRNA were analyzed by Real-time PCR while distribution of NHE-1, 2 and 4 protein were observed by immunohistochemistry. RESULTS: Treatment with 50 and 100 mg/kg/day genistein caused marked increase in the levels of expression and distribution of NHE-1, 2 and 4 proteins in the endocervical epithelia. Levels of Nhe-1, 2 and 4 mRNA in the cervix were also increased. Coadministration of ICI 182 780 and genistein reduced the expression levels of NHE-1, 2 and 4 proteins and mRNAs in the cervix. CONCLUSIONS: Enhanced expression of NHE-1, 2 and 4 proteins and mRNAs expression in cervix under genistein influence could help to restore the cervicovaginal fluid pH that might help to prevent cervicovaginal complications related to menopause.

Sex-steroid regulation of relaxin receptor isoforms (RXFP1 & RXFP2) expression in the patellar tendon and lateral collateral ligament of female WKY rats.

UNLABELLED: The incidence of non-contact knee injury was found higher in female than in male and is related to the phases of the menstrual cycle. This raised the possibility that female sex-steroids are involved in the mechanism underlying this injury via affecting the expression of the receptors for relaxin, a peptide hormone known to modulate ligament laxity. Therefore, this study aims to investigate the effect of sex-steroids on relaxin receptor isoforms (RXFP1 & RXFP2) expression in the ligaments and tendons of the knee. METHODS: Ovariectomized adult female WKY rats were treated with different doses of estrogen (0.2, 2, 20 µg/kg), progesterone (4mg) and testosterone (125 & 250µg/kg) for three consecutive days. At the end of the treatment, the animals were sacrificed and the patellar tendon and lateral collateral ligament were harvested for mRNA and protein expression analyses by Real Time PCR and Western blotting respectively. RESULTS: RXFP1, the main isoform expressed in these knee structures and RXFP2 showed a dose-dependent increase in expression with estrogen. Progesterone treatment resulted in an increase while testosterone caused a dose-dependent decrease in the mRNA and protein expression of both relaxin receptor isoforms. DISCUSSION: Progesterone and high dose estrogen up-regulate while testosterone down-regulates RXFP1 and RXFP2 expression in the patellar tendon and lateral collateral ligament of rat's knee. CONCLUSION: Relaxin receptor isoforms up-regulation by progesterone and high dose estrogen could provide the basis for the reported increase in knee laxity while down-regulation of these receptor isoforms by testosterone could explain low incidence of non-contact knee injury in male.

Chlorophytum borivilianum root extract maintains near normal blood glucose, insulin and lipid profile levels and prevents oxidative stress in the pancreas of streptozotocin-induced adult male diabetic rats.

UNLABELLED: The effect of C. borivilianum root on blood glucose, glycated hemoglobin (HbAIc), insulin and lipid profile levels in diabetes mellitus are not fully understood. This study therefore investigated the effect of C. borivilianum root on the above parameters and oxidative stress of the pancreas in diabetes. METHODS: C. borivilianum root aqueous extract (250 and 500 mg/kg/day) was administered to streptozotocin (STZ)-induced male diabetic rats for 28 days. Body weight, blood glucose, HbA1c, insulin, lipid profile levels and glucose homeostasis indices were determined. Histopathological changes and oxidative stress parameters i.e. lipid peroxidation (LPO) and antioxidant enzymes activity levels of the pancreas were investigated. RESULTS: C. borivilianum root extract treatment to diabetic rats maintained near normal body weight, blood glucose, HbA1c, lipid profile and insulin levels with higher HOMA-ß cell functioning index, number of Islets/pancreas, number of ß-cells/Islets however with lower HOMA-insulin resistance (IR) index as compared to non-treated diabetic rats. Negative correlations between serum insulin and blood glucose, HbA1c, triglyceride (TG) and total cholesterol (TC) levels were observed. C. borivilianum root extract administration prevented the increase in lipid peroxidation and the decrease in activity levels of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) with mild histopathological changes in the pancreas of diabetic rats. CONCLUSIONS: C. borivilianum root maintains near normal levels of these metabolites and prevented oxidative stress-induced damage to the pancreas in diabetes.

Chlorophytum borivilianum (Safed Musli) root extract prevents impairment in characteristics and elevation of oxidative stress in sperm of streptozotocin-induced adult male diabetic Wistar rats.

BACKGROUND: We hypothesized that C. borivilianum root, known to improve male reproductive performance, prevents impairment in characteristics, morphology and elevation of oxidative stress in sperm of diabetics. We therefore investigated the effect of aqueous root extract of C. borivilianum on these parameters in diabetic rat model. METHODS: C. borivilianum root aqueous extract (250 and 500 mg/kg/day) or glibenclamide (600 µg/kg/day) were administered to streptozotocin (STZ)-induced diabetic male rats for 28 consecutive days. At the end of treatment, animals were sacrificed and sperm were collected. Sperm count and percentages of forward motility, viability, hypoosmotic swelling (HOS) tail-coiled and abnormal sperm were evaluated. Sperm lipid peroxidation product (LPO), hydrogen peroxide (H2O2) and nitric oxide (NO) levels, total antioxidant capacity (TAC), activity levels of endogenous antioxidant enzymes (superoxide dismutase-SOD, catalase-CAT and glutathione peroxidase-GPx), epididymal sperm density, serum fasting blood glucose (FBG) and HbA1c levels were measured. The expression of sperm caspase-3 was assessed. Meanwhile, in-vitro free radical scavenging activity of C. borivilianum root extract was determined and the root extract was analyzed for the presence of bioactive compounds by FTIR spectroscopy. RESULTS: C. borivilianum root aqueous extract prevents the decrease in sperm count, percentages of forward motility, viability, HOS and the increase in abnormal sperm percentage and caspase-3 level in diabetic rats. Sperm LPO, H2O2 and NO levels, FBG and HbA1c were lower while TAC, SOD, CAT, GPx and epididymal sperm density were higher in diabetic rats receiving C. borivilianum root extract treatment. C. borivilianum root exhibited strong in-vitro free radical scavenging activity which may be due to the phenolic compound. CONCLUSIONS: C. borivilianum root extract prevents impairment in sperm characteristics and morphology via preventing elevation of oxidative stress, apoptosis and free radicals levels of the sperm in diabetes. These effects may be achieved through maintaining sperm antioxidant level which could be related to free radical scavenging activity of the root extract by phenolic compounds. These effects could also be due to ability of the extract to maintain near normal serum FBG and HBA1c levels in diabetes.

Diverse roles of prostaglandins in blastocyst implantation.

Prostaglandins (PGs), derivatives of arachidonic acid, play an indispensable role in embryo implantation. PGs have been reported to participate in the increase in vascular permeability, stromal decidualization, blastocyst growth and development, leukocyte recruitment, embryo transport, trophoblast invasion, and extracellular matrix remodeling during implantation. Deranged PGs syntheses and actions will result in implantation failure. This review summarizes up-to-date literatures on the role of PGs in blastocyst implantation which could provide a broad perspective to guide further research in this field.

Leukemia inhibitory factor: roles in embryo implantation and in nonhormonal contraception.

Leukaemia inhibitory factor (LIF) plays an indispensible role in embryo implantation. Aberrant LIF production is linked to implantation failure. LIF regulates multiple processes prior to and during implantation such as uterine transformation into a receptive state, decidualization, blastocyst growth and development, embryo-endometrial interaction, trophoblast invasion, and immune modulation. Due to its critical role, LIF has been a target for a nonhormonal contraception. In this review, we summarize up-to-date information on the role of LIF in implantation and its role in contraception.

Testosterone reduces knee passive range of motion and expression of relaxin receptor isoforms via 5α-dihydrotestosterone and androgen receptor binding.

Ovarian steroids such as estrogen and progesterone have been reported to influence knee laxity. The effect of testosterone, however, remains unknown. This study investigated the effect of testosterone on the knee range of motion (ROM) and the molecular mechanisms that might involve changes in the expression of relaxin receptor isoforms, Rxfp1 and Rxfp2 in the patella tendon and lateral collateral ligament of the female rat knee. Ovariectomized adult female Wistar rats received three days treatment with peanut oil (control), testosterone (125 and 250 µg/kg) and testosterone (125 and 250 µg/kg) plus flutamide, an androgen receptor blocker or finasteride, a 5α-reductase inhibitor. Duplicate groups received similar treatment however in the presence of relaxin (25 ng/kg). A day after the last drug injection, knee passive ROM was measured by using a digital miniature goniometer. Both tendon and ligament were harvested and then analysed for protein and mRNA expression for Rxfp1 and Rxfp2 respectively. Knee passive ROM, Rxfp1 and Rxfp2 expression were significantly reduced following treatment with testosterone. Flutamide or finasteride administration antagonized the testosterone effect. Concomitant administration of testosterone and relaxin did not result in a significant change in knee ROM as compared to testosterone only treatment; however this was significantly increased following flutamide or finasteride addition. Testosterone effect on knee passive ROM is likely mediated via dihydro-testosterone (DHT), and involves downregulation of Rxfp1 and Rxfp2 expression, which may provide the mechanism underlying testosterone-induced decrease in female knee laxity.

Genistein induces increase in fluid pH, Na+ and HCO3(-) concentration, SLC26A6 and SLC4A4 (NBCe1)-B expression in the uteri of ovariectomized rats.

Genistein has been reported to stimulate luminal HCO3(-) secretion. We hypothesized that genistein mediates this effect via SLC26A6 and SLC4A4 (NBCe1) transporters. Our study aimed to: investigate changes in uterine fluid pH, Na+ and HCO3(-) concentration and expression of uterine SLC26A6 and NBCe1 under genistein effect. Ovariectomized adult female rats received 25, 50 and 100 mg/kg/day genistein for a week with and without ICI 182780. A day after the last injection, in vivo uterine perfusion was performed to collect uterine fluid for Na+, HCO3(-) and pH determination. The animals were then sacrificed and uteri were removed for mRNA and protein expression analyses. SLC26A6 and NBCe1-A and NBCe1-B distribution were visualized by immunohistochemistry (IHC). Genistein at 50 and 100 mg/kg/day stimulates uterine fluid pH, Na+ and HCO3(-) concentration increase. Genistein at 100 mg/kg/day up-regulates the expression of SLC26A6 and SLC4A4 mRNA, which were reduced following concomitant ICI 182780 administration. In parallel, SLC26A6 and NBCe1-B protein expression were also increased following high dose genistein treatment and were localized mainly at the apical membrane of the luminal epithelia. SLC26A6 and NBCe1-B up-regulation by genistein could be responsible for the observed increase in the uterine fluid pH, Na+ and HCO3(-) concentration under this condition.

Marantodes pumilum (Kacip Fatimah) Aqueous Extract Enhances Osteoblast and Suppresses Osteoclast Activities in Cancellous Bone of a Rat Model of Postmenopause.

Evidence pointed towards the benefits of Marantodes pumilum in treating osteoporosis after menopause; however, the detailed mechanisms still have not been explored. Therefore, this study aims to identify the molecular mechanisms underlying M. pumilum's bone-protective effect via the involvement of RANK/RANKL/OPG and Wnt/ß-catenin signaling pathways. Ovariectomized adult female rats were given M. pumilum leaf aqueous extract (MPLA) (50 and 100 mg/kg/day) and estrogen (positive control) orally for twenty-eight consecutive days. Following the treatment, rats were sacrificed, and femur bones were harvested. Blood was withdrawn for analysis of serum Ca2+, PO43-, and bone alkaline phosphatase (BALP) levels. The bone microarchitectural changes were observed by H&E and PAS staining and distribution and expression of RANK/RANKL/OPG and Wnt3a/ß-catenin and its downstream proteins were determined by immunohistochemistry, immunofluorescence, Western blot, and real-time PCR. MPLA treatment increased serum Ca2+ and PO43- levels and reduced serum BALP levels (p < 0.05). Besides, deterioration in cancellous bone microarchitecture and the loss of bone glycogen and collagen content were mitigated by MPLA treatment. Levels of RANKL, Traf6, and NF-kB but not RANK in bone were decreased; however, levels of OPG, Wnt3a, LRP-5, Frizzled, Dvl, ß-catenin, RUNX, and Bmp-2 in bone were increased following treatment with MPLA. In conclusion, MPLA helps to protect against bone deterioration in estrogen deficiency state and thus, this herb could potentially be used to ameliorate osteoporosis in women after menopause.

In-vivo functional study on the involvement of CFTR, SLC26A6, NHE-1 and CA isoenzymes II and XII in uterine fluid pH, volume and electrolyte regulation in rats under different sex-steroid influence.

UNLABELLED: Precise control of uterine fluid pH, volume and electrolytes is important for the reproductive processes. In this study, we examined the functional involvement of multiple proteins including Cystic Fibrosis Transmembrane Regulator (CFTR), Cl(-)/HCO3 (-) exchanger (SLC26A6), sodium-hydrogen exchanger-1 (NHE-1) and carbonic anhydrase (CA) in the regulation of these uterine fluid parameters. METHODS: Adult female WKY rats were divided into intact, non-ovariectomised at different oestrous cycle phases and ovariectomised treated with sex-steroids. Following oestrous phase identification or sex-steroid treatment, in-vivo uterine perfusion was performed with and without the presence of these inhibitors: glibenclamide, DIDS, ACTZ and EIPA. The pH, volume, Cl(-), HCO3 (-) and Na(+) concentrations of the perfusate from different groups were then analyzed. Meanwhile, the expression of CFTR, SLC26A6, NHE-1, CAII and CAXII was visualized by immunohistochemistry (IHC). RESULTS: Parallel increase in the pH, volume, Cl(-), HCO3 (-) and Na(+) concentrations was observed at estrus (Es), proestrus (Ps) and following 17ß-oestradiol (E) treatment, which was inhibited by glibenclamide, DIDS and ACTZ while parallel reduction in these parameters was observed at diestrus (Ds) and following progesterone (P) treatment which was inhibited by ACTZ and EIPA. CFTR and SLC26A6 expression were up-regulated under E dominance, while NHE-1 expression was up-regulated under P dominance. Meanwhile, CA isoenzymes were expressed under both E and P influence. CONCLUSION: CFTR, SLC26A6 and CA were involved in mediating parallel increase in the uterine fluid volume, pH and electrolyte concentration under E while NHE and CA were involved in mediating the reduction of these parameters under P.