Reproductive Biology and Endocrinology - Latest articles

Opposing effects of D-aspartic acid and nitric oxide on tuning of testosterone production in mallard testis during the reproductive cycle

Maria M Di Fiore, Claudia Lamanna, Loredana Assisi and Virgilio Botte — 2008-07-04

Background: D-Aspartic acid (D-Asp) and nitric oxide (NO) play an important role in tuning testosterone production in the gonads of male vertebrates. In particular, D-Asp promotes either the synthesis or the release of testosterone, whereas NO inhibits it. In this study, we have investigated for the first time in birds the putative effects of D-Asp and NO on testicular testosterone production in relation to two phases of the reproductive cycle of the adult captive wild-strain mallard (Anas platyrhynchos) drake. It is a typical seasonal breeder and its cycle consists of a short reproductive period (RP) in the spring (April-May) and a non reproductive period (NRP) in the summer (July), a time when the gonads are quiescent. The presence and the localization of D-Asp and NO in the testis and the trends of D-Asp, NO and testosterone levels were assessed during the main phases of the bird's reproductive cycle. Furthermore, in vitro experiments revealed the direct effect of exogenously administered D-Asp and NO on testosterone steroidogenesis. Methods: By using immunohistochemical (IHC) techniques, we studied the presence and the distributional pattern of D-Asp and NO in the testes of RP and NRP drakes. D-Asp levels were evaluated by an enzymatic method, whereas NO content, via nitrite, was assessed using biochemical measurements. Finally, immunoenzymatic techniques determined testicular testosterone levels. Results: IHC analyses revealed the presence of D-Asp and NO in Leydig cells. The distributional pattern of both molecules was in some way correlated to the steroidogenic pathway, which is involved in autocrine testosterone production. Indeed, whereas NO was present only during the NRP, D-Asp was almost exclusively present during the RP. Consistently, the high testosterone testicular content occurring during RP was coupled to a high D-Asp level and a low NO content in the gonad. By contrast, in sexually inactive drakes (NRP), the low testosterone content in the gonad was coupled to a low D-Asp content and to a relatively high NO level. Consequently, to determine the exogenous effects of the two amino acids on testosterone synthesis, we carried out in vitro experiments using testis sections deriving from both the RP and NRP. When testis slices were incubated for 60 or 120 min with D-Asp, testosterone was enhanced, whereas in the presence of L-Arg, a precursor of NO, it was inhibited. Conclusions: Our results provide new insights into the involvement of D-Asp and NO in testicular testosterone production in the adult captive wild-strain mallard drake. The localization of these two molecules in the Leydig cells in different periods of the reproductive cycle demonstrates that they play a potential role in regulating local testosterone production.

Differential transferrin expression in placentae from normal and abnormal pregnancies: a pilot study

2008-07-02

Background: The placenta is an important site for iron metabolism in humans. It transfers iron from the mother to the fetus. One of the major iron transport proteins is transferrin, which is a blood plasma protein crucial for iron uptake. Its localization and expression may be one of the markers to distinguish placental dysfunction. Methods: In the experimental study we used antibody preparation, mass spectrometric analysis, biochemical and immunocytochemical methods for characterization of transferrin expression on the human choriocarcinoma cell line JAR (JAR cells), placental lysates, and cryostat sections. Newly designed monoclonal antibody TRO-tf-01 to human transferrin was applied on human placentae from normal (n=3) and abnormal (n=9) pregnancies. Results: Variations of transferrin expression were detected in villous syncytiotrophoblast, which is in direct contact with maternal blood. In placentae from normal pregnancies, the expression of transferrin in the syncytium was significantly lower (p<0.001) when compared to placentae from abnormal ones (gestational diabetes, pregnancy induced hypertension, drug abuse). Conclusions: These observations suggest that in the case of abnormal pregnancies, the fetus may require higher levels of transferrin in order to prevent iron depletion due to the stress from the placental dysfunction.

Risk assessment does not explain high prevalence of gestational diabetes mellitus in a large group of Sardinian women

2008-07-02

Background: A very high prevalence (22.3%) of gestational diabetes mellitus (GDM) was recently reported following our study on a large group of Sardinian women. In order to explain such a high prevalence we sought to characterise our obstetric population through the analysis of risk factors and their association with the development of GDM. Methods: The prevalence of risk factors and their association with the development of GDM were evaluated in 1103 pregnancies (247 GDM and 856 control women). The association of risk factors with GDM was calculated according to logistic regression. Sensitivity and specificity of risk assessment strategy were also calculated. Results: None of the risk factors evaluated showed an elevated frequency in our population. The high risk patients were 231 (20.9%). Factors with a stronger association with GDM development were obesity (OR 3.7, 95% CI 2.08-6.8), prior GDM (OR 3.1, 95% CI 1.69-5.69), and family history of Type 2 diabetes (OR 2.6, 95% CI 1.81-3.86). Only patients over 35 years of age were more represented in the GDM group (38.2% vs 22.6% in the non-GDM cases, P<0.001). Type 2 diabetes in second-degree relatives was equally represented in GDM and non-GDM subjects, while prior poor obstetrical outcomes mostly characterized non-GDM women (17.5% vs 10.6%, P<0.001). The "average risk" assessment better characterized non-GDM patients (76.8% vs 57.8%, P<0.001). The logistic regression analysis confirmed that Type 2 diabetes in second-degree relatives, prior poor obstetrical outcomes and the "average risk" definition did not predict the development of GDM. Conclusion: Such a high prevalence of GDM in our population does not seem to be related to the abnormal presence of some known risk factors, and appears in contrast with the prevalence of Type 2 diabetes in Sardinia. Further studies are needed to explain the cause such a high prevalence of GDM in Sardinia. The "average risk" definition is not adequate to predict GDM in our population.

Expression profiles for six zebrafish genes during gonadal sex differentiation

2008-06-30

Background: The mechanism of sex determination in zebrafish is largely unknown and neither sex chromosomes nor a sex-determining gene have been identified. This indicates that sex determination in zebrafish is mediated by genetic signals from autosomal genes. The aim of this study was to determine the precise timing of expression of six genes previously suggested to be associated with sex differentiation in zebrafish. The current study investigates the expression of all six genes in the same individual fish with extensive sampling dates during sex determination and -differentiation. Results: In the present study, we have used quantitative real-time PCR to investigate the expression of ar, sox9a, dmrt1, fig alpha, cyp19a1a and cyp19a1b during the expected sex determination and gonadal sex differentiation period. The expression of the genes expected to be high in males (ar, sox9a and dmrt1a) and high in females (fig alpha and cyp19a1a) was segregated in two groups with more than 10 times difference in expression levels. All of the investigated genes showed peaks in expression levels during the time of sex determination and gonadal sex differentiation. Expression of all genes was investigated on cDNA from the same fish allowing comparison of the high and low expressers of genes that are expected to be highest expressed in either males or females. There were 78% high or low expressers of all three "male" genes (ar, sox9a and dmrt1) in the investigated period and 81% were high or low expressers of both "female" genes (fig alpha and cyp19a1a). When comparing all five genes with expected sex related expression 56% show expression expected for either male or female. Furthermore, the expression of all genes was investigated in different tissue of adult male and female zebrafish. Conclusion: In zebrafish, the first significant peak in gene expression during the investigated period (2-40 dph) was dmrt1 at 10 dph which indicates involvement of this gene in the early gonadal sex differentiation of males.

Autoimmunity in gestational diabetes mellitus in Sardinia: a preliminary case-control report

2008-06-29

Background: We previously reported a high prevalence (22.3%) of gestational diabetes mellitus (GDM) in a large group of Sardinian women, in contrast with the prevalence of Type 2 diabetes. Sardinia has an unusual distribution of haplotypes and genotypes, with the highest population frequency of HLA DR3 in the world, and after Finland, the highest prevalence of Type 1 diabetes and Autoimmune-related Diseases. In this study we preliminarily tested the prevalence of serological markers of Type 1 diabetes in a group of Sardinian GDM patients. Methods: We determined glutamic decarboxylase antibodies (anti-GAD65), protein tyrosine phosphatase ICA 512 (IA2) antibodies (anti-IA2), and IAA in 62 GDM patients, and in 56 controls with matching age, gestational age and parity. Results: We found a high prevalence and very unusual distribution of antibodies in GDM patients (38.8%), the anti-IA2 being the most frequent antibody. Out of all our GDM patients, 38.8% (24 of 62) were positive for at least one antibody. Anti-IA2 was present in 29.0 % (18 out of 62) vs. 7.1% (4 out of 56) in the controls (P<0.001). IAA was present in 14.5% (9 out of 62) of our GDM patients, and absent in the control subjects (P<0.001). Anti-GAD65 was also present in GDM patients, with a prevalence of 3.2% (2 out of 62) while it was absent in the control group (P=NS). Pre-gestational weight was significantly lower (57.78 +/- 9.8 vs 65.9 +/- 17.3 P=0.04) in auto-antibodies- positive GDM patients. Conclusions: These results are in contrast with the very low prevalence of all antibodies reported in Italy. If confirmed, they could indicate that a large proportion of GDM patients in Sardinia have an autoimmune origin, in accordance with the high prevalence of Type 1 diabetes.

Treatment of pregnant rats with oleoyl-estrone slows down pup fat deposition after weaning

Beatriz Garcia-Pelaez, Ruth Vila and Xavier Remesar — 2008-06-20

Background: In rats, oral oleoyl-estrone (OE) decreases food intake and body lipid content. The aim of this study was to determine whether OE treatment affects the energy metabolism of pregnant rats and eventually, of their pups; i.e. changes in normal growth patterns and the onset of obesity after weaning. Methods: Pregnant Wistar rats were treated with daily intragastric gavages of OE in 0.2 ml sunflower oil from days 11 to 21 of pregnancy (i.e. 10 nmol oleoyl-estrone/g/day). Control animals received only the vehicle. Plasma and hormone metabolites were determined together with variations in cellularity of adipose tissue. Results: Treatment decreased food intake and lowered weight gain during late pregnancy, mainly because of reduced adipose tissue accumulation in different sites. OE-treated pregnant rats' metabolic pattern after delivery was similar to that of controls. Neonates from OE-treated rats weighed the same as those from controls. They also maintained the same growth rate up to weaning, but pups from OE-treated rats slowed their growth rate afterwards, despite only limited differences in metabolite concentrations. Conclusions: The OE influences on pup growth can be partially buffered by maternal lipid mobilization during the second half of pregnancy. This maternal metabolic "imprinting" may condition the eventual accumulation of adipose tissue after weaning, and its effects can affect the regulation of body weight up to adulthood.

HTRA3 expression in non-pregnant rhesus monkey ovary and endometrium, and at the maternal-fetal interface during early pregnancy

2008-06-18

Background: HTRA3 is a recently identified member of the mammalian serine protease family HTRA (high temperature requirement factor A). In both the rodent and the human HTRA3 is transcribed into two mRNA species (long and short) through alternative splicing. We have previously shown that HTRA3 is expressed in the mature rat ovary and may be involved in folliculogenesis and luteinisation. HTRA3 is also upregulated during mouse and human placental development. The current study investigated whether HTRA3 is also localised in the primate ovary (rhesus monkey n = 7). In addition, we examined the non-pregnant rhesus monkey endometrium (n = 4) and maternal-fetal interface during early pregnancy (n = 5) to further investigate expression of HTRA3 in primate endometrium and placentation. Methods: HTRA3 mRNA levels in several rhesus monkey tissues was determined by semiquantitative RT-PCR. Protein expression and localisation of HTRA3 was determined by immunohistochemistry. Results: Long and short forms of HTRA3 mRNA were detected in the ovary, aorta, bladder, small intestine, skeletal muscle, heart and uterus but not the liver nor the kidney. HTRA3 protein was immunolocalised to the oocyte of all follicular stages in the rhesus monkey ovary. Protein expression in mural and cumulus granulosa cells of late secondary follicles increased significantly compared to granulosa cells of primordial, primary and secondary follicles. Mural and cumulus granulosa cells of antral follicles also showed a significant increase in expression. Staining intensity was higher in the granulosa-lutein cells compared to the theca-lutein cells of corpora lutea (n = 3). In the non-pregnant monkey endometrium, HTRA3 was detected in the glandular epithelium. The basalis endometrial glands showed higher staining intensity than functionalis endometrial glands. During early pregnancy, strong staining for HTRA3 protein was seen in both maternal decidual cells and glands. Conclusion: We propose that HTRA3 may be involved in folliculogenesis and luteinisation in the primate ovary. Furthermore, similar to previous findings in the human, HTRA3 is possibly a factor involved in and potentially important for primate placentation.

Nutritional skewing of conceptus sex in sheep: effects of a maternal diet enriched in rumen-protected polyunsaturated fatty acids (PUFA)

2008-06-09

Background: Evolutionary theory suggests that in polygynous mammalian species females in better body condition should produce more sons than daughters. Few controlled studies have however tested this hypothesis and controversy exists as to whether body condition score or maternal diet is in fact the determining factor of offspring sex. Here, we examined whether maternal diet, specifically increased n-6 polyunsaturated fatty acid (PUFA) intake, of ewes with a constant body condition score around the time of conception influenced sex ratio. Methods: Ewes (n = 44) maintained in similar body condition throughout the study were assigned either a control (C) diet or one (F) enriched in rumen-protected PUFA, but otherwise essentially equivalent, from four weeks prior to breeding until d13 post-estrus. On d13, conceptuses were recovered, measured, cultured to assess their capacity for interferon-tau (IFNT) production and their sex determined. The experiment was repeated with all ewes being fed the F diet to remove any effects of parity order on sex ratio. Maternal body condition score (BCS), plasma hormone and metabolite concentrations were also assessed throughout the study and related to diet. Results: In total 129 conceptuses were recovered. Ewes on the F diet produced significantly more male than female conceptuses (proportion male = 0.69; deviation from expected ratio of 0.5, P < 0.001). Conceptus IFNT production was unaffected by diet (P > 0.1), but positively correlated with maternal body condition score (P < 0.05), and was higher (P < 0.05) in female than male conceptuses after 4 h culture. Maternal plasma hormone and metabolite concentrations, especially progesterone and fatty acid, were also modulated by diet. Conclusion: These results provide evidence that maternal diet, in the form of increased amounts of rumen-protected PUFA fed around conception, rather than maternal body condition, can skew the sex ratio towards males. These observations may have implications to the livestock industry and animal management policies when offspring of one sex may be preferred over the other.

In infertile women, cells from Chlamydia trachomatis infected site release higher levels of interferon-gamma, interleukin-10 and tumor necrosis factor-alpha upon heat shock protein stimulation than fertile women

Pragya Srivastava, Rajneesh Jha, Sylvette Bas, Sudha Salhan and Aruna Mittal — 2008-05-20

Background: The magnitude of reproductive morbidity associated with sexually transmitted Chlamydia trachomatis infection is enormous. Association of antibodies to chlamydial heat shock proteins (cHSP) 60 and 10 with various disease sequelae such as infertility or ectopic pregnancy has been reported. Cell-mediated immunity is essential in resolution and in protection to Chlamydia as well as is involved in the immunopathogenesis of chlamydial diseases. To date only peripheral cell mediated immune responses have been evaluated for cHSP60. These studies suggest cHSPs as important factors involved in immunopathological condition associated with infection. Hence study of specific cytokine responses of mononuclear cells from the infectious site to cHSP60 and cHSP10 may elucidate their actual role in the cause of immunopathogenesis and the disease outcome. Methods: Female patients (n = 368) attending the gynecology out patient department of Safdarjung hospital, New Delhi were enrolled for the study and were clinically characterized into two groups; chlamydia positive fertile women (n = 63) and chlamydia positive infertile women (n = 70). Uninfected healthy women with no infertility problem were enrolled as controls (n = 39). cHSP60 and cHSP10 specific cytokine responses (Interferon (IFN)-gamma, Interleukin (IL)-10, Tumor Necrosis Factor (TNF)-alpha, IL-13 and IL-4) were assessed by ELISA in stimulated cervical mononuclear cell supernatants. Results: cHSP60 and cHSP10 stimulation results in significant increase in IFN-gamma (P = 0.006 and P = 0.04 respectively) and IL-10 levels (P = 0.04) in infertile group as compared to fertile group. A significant cHSP60 specific increase in TNF-alpha levels (P = 0.0008) was observed in infertile group as compared to fertile group. cHSP60 and cHSP10 specific IFN-gamma and IL-10 levels were significantly correlated (P < 0.0001, r = 0.54 and P = 0.004, r = 0.33 respectively) in infertile group. Conclusion: Our results suggest that exposure to chlamydial heat shock proteins (cHSP60 and cHSP10) could significantly affect mucosal immune function by increasing the release of IFN-gamma, IL-10 and TNF-alpha by cervical mononuclear cells.

Plasma 11-deoxycorticosterone (DOC) and mineralocorticoid receptor testicular expression during rainbow trout Oncorhynchus mykiss spermiation: implication with 17alpha, 20beta-dihydroxyprogesterone on the milt fluidity?

2008-05-19

Background: In rainbow trout (Oncorhynchus mykiss), the endocrine control of spermiation is not fully understood. Besides 11ketotestosterone (11KT) and 17alpha, 20beta-dihydroxyprogesterone (MIS), the potential physiological ligand of the mineralocorticoid receptor (MR) 11-deoxycorticosterone (DOC), is a credible candidate in O. mykiss spermiation regulation as spermiation is accompanied with changes in aqueous and ionic flows. Methods: In this study, we investigated potential roles of DOC during spermiation 1) by describing changes in blood plasma DOC level, MR mRNA abundance during the reproductive cycle and MR localization in the reproductive tract 2) by investigating and comparing the effects of DOC (10 mg/kg) and MIS (5 mg/kg) supplementations on sperm parameters 3) by measuring the in vitro effect of DOC on testis MIS production. Results: The plasma concentration of DOC increased rapidly at the end of the reproductive cycle to reach levels that were 10–50 fold higher in mature males than in immature fish. MR mRNA relative abundance was lower in maturing testes when compared to immature testes, but increased rapidly during the spermiation period, immediately after the plasma rise in DOC. At this stage, immunohistochemistry localized MR protein to cells situated at the periphery of the seminiferous tubules and in the efferent ducts. Neither DOC nor MIS had significant effects on the mean sperm volume, although MIS treatment significantly increased the percentage of males producing milt. However, a significant reduction in the spermatocrit was observed when DOC and MIS were administrated together. Finally, we detected an inhibitory effect of DOC on testis MIS production in vitro. Conclusion: These results are in agreement with potential roles of DOC and MR during spermiation and support the hypothesis that DOC and MIS mechanisms of action are linked during this reproductive stage, maybe controlling milt fluidity. They also confirm that in O. mykiss MIS is involved in spermiation induction.