Reproductive Biology and Endocrinology - Latest Articles

An imbalance between apoptosis and proliferation contributes to follicular persistence in polycystic ovaries in rats

Natalia Salvetti — 2009-07-01T00:00:00Z

Background: Cystic ovarian disease is an important cause of infertility that affects bovine, ovine, caprine and porcine species and even human beings. Alterations in the ovarian micro-environment of females with follicular cysts could alter the normal processes of proliferation and programmed cell death in ovarian cells. Thus, our objective was to evaluate apoptosis and proliferation in ovarian cystic follicles in rats in order to investigate the cause of cystic follicle formation and persistence. Methods: We compared the number of in situ apoptotic cells by TUNEL assay, expression of active caspase-3 and members of Bcl-2 family by immunohistochemistry; and cell proliferation by the expression of the proliferation markers: PCNA and Ki-67. Results: The proliferation index was low in granulosa of tertiary and cystic follicles of light exposed rats when compared with tertiary follicles of control animals, while in theca interna only cystic follicles presented low proliferation index when compared with tertiary follicles (p<0.05). The granulosa of cysts exhibited a similar cell DNA fragmentation to early atretic follicles. In the granulosa and theca interna, active caspase-3 shown similar immunostaining levels in tertiary and cystic follicles (p<0.05). The granulosa cells presented high expression of Bcl-2, Bcl-xL and Bcl-w in the tertiary and cystic follicles with diminishing intensity in the atretic follicles, except with Bcl-w where the intensity was maintained in the atretic follicles (p<0.05). The expression of Bax was weak in the healthy and cystic follicles. In the theca interna, Bcl-2 expression was the same as the pattern found in the granulosa; no differences were found between tertiary and cystic follicles from both groups for Bcl-xL and Bcl-w. The expression of Bax in this layer was higher in the tertiary follicles of the treated animals (p<0.05) while the values for cystic follicles were similar to those in the tertiary follicles of controls. The theca externa showed low expression of the pro and anti-apoptotic proteins. Conclusion: These results show that the combination of weak proliferation indices and low apoptosis observed in follicular cysts, could explain the cause of the slow growth of cystic follicles and the maintenance of a static condition without degeneration, which leads to their persistence. These alterations may be due to structural and functional modifications that take place in these cells and could be related to hormonal changes in animals with this condition.

Seminiferous tubule transfection in vitro to define post-meiotic gene regulation

Sandra Danner — 2009-06-29T00:00:00Z

Background: Post-meiotically expressed genes in the testis are essential for the proper progression of spermatogenesis, and yet, aside from the construction of individual transgenic mice using specific promoters to drive reporter plasmids, there are only very limited possibilities for relevant and quantitative analysis of gene promoters. This is due to the special nature of post-meiotic haploid cells, which to date are not represented in any appropriate cell-lines. This article reports the development of novel methodology using isolated and cultured rat seminiferous tubules in a multiwell format, into which promoter-reporter constructs can be introduced by a combination of microinjection and electroporation. Methods: Culture conditions were developed which allowed the continued incubation of isolated rat seminiferous tubules for up to 48h without obvious cell death and loss of post-meiotic cells. Transfection of intact seminiferous tubules by microinjection and electroporation was optimized to achieve high expression efficiencies of control plasmids, using either fluorescent protein or luciferase as reporters, thereby allowing both morphological as well as quantitative assessment. Results: Successful transfection was achieved into all cell types except for mature spermatozoa. However, there appeared to be only limited cell-type specificity for the promoters used, even though these had appeared to be specific when used in transgenic animals. Conclusion: We have devised a methodology which allows relatively high throughput analysis of post-meiotic gene promoters into primary cells of intact seminiferous tubules. An apparent lack of cell-type specificity suggests that the gene fragments used do not contain sufficient targeting information, or that the transient episomal expression of the constructs does not encourage appropriate expression specificity. The results also highlight the doubtful interpretation of many studies using heterologous transfection systems to analyse post-meiotically expressed genes.

Activin A induces ovine follicle stimulating hormone beta using -169/-58 bp of its promoter and a simple TATA box

Sang-oh Han — 2009-06-24T00:00:00Z

Background: Activin A increases production of follicle stimulating hormone (FSH) by inducing transcription of its beta subunit (FSHB). This induction has been studied here in LbetaT2 gonadotropes using transient expression of ovine FSHBLuc (-4741 bp of ovine FSHB promoter plus exon/intron 1 linked to Luc). Several sequences between -169/-58 bp of the ovine FSHB proximal promoter are necessary for induction by activin A in LbetaT2 cells, but deletions between -4741/-752 bp decrease induction > 70% suggesting the existence of other important 5' sequences. Induction disappears if a minimal T81 thymidine kinase promoter replaces the ovine FSHB TATA box and 3' exon/intron. The study reported here was designed to determine if sequences outside -169/-58 bp are important for induction of ovine FSHB by activin A. Methods: Progressively longer deletions of ovine FSHBLuc were created between -4741/-195 bp. Deletions internal to this region were created also, but replaced with substitute DNA. The ovine FSHB TATA box region (-40/+3 bp) was replaced by thymidine kinase and rat prolactin minimal promoters, and substitutions were made in 3' intron/exon sequences. All constructs were tested for basal and activin A-induced expression in LbetaT2 cells. Results: Successive 5' deletions progressively lowered fold-induction by activin A from 9.5 to zero, but progressively increased basal expression. Replacing deletions with substitute DNA showed no changes in basal expression or fold-induction. Induction by activin A was supported by the minimal rat prolactin promoter (TATA box) but not the thymidine kinase promoter (no TATA box). Replacement mutations in the 3' region did not decrease induction by activin A. Conclusions: The data show that specific ovine FSHB sequences 5' to -175 bp or 3' of the transcription start site are not required for induction by activin A. A minimal TATA box promoter supports induction by activin A, but the sequence between the TATA box and transcription start site seems unimportant.

Chronic crude garlic-feeding modified adult male rat testicular markers: mechanisms of action

Imen Hammami — 2009-06-24T00:00:00Z

Background: Garlic or Allium sativum (As) shows therapeutic effects such as reduction of blood pressure or hypercholesterolemia but side-effects on reproductive functions remain poorly investigated. Because of garlic's chemical complexity, the processing methods and yield in preparations differ in efficacy and safety. In this context, we clarify the mechanisms of action of crushed crude garlic on testicular markers. Methods: During one month of treatment, 24 male rats were fed 5%, 10% and 15% crude garlic. Results: We showed that crude garlic-feeding induced apoptosis in testicular germ cells (spermatocytes and spermatids). This cell death process was characterized by increased levels of active CASP3 but not CASP6. Expression of the caspase inhibitors BIRC3 and BIRC2 was increased at all doses of As while expression of XIAP and BIRC5 was unchanged. Moreover, expression of the IAP inhibitor DIABLO was increased at doses 10% and 15% of As. The germ cell death process induced by As might be related to a decrease in testosterone production because of the reduced expression of steroidogenic enzymes (Star, Cyp11a, Hsd3b5 and Hsd17b). Evaluation of Sertoli markers showed that TUBB3 and GSTA2 expression was unchanged. In contrast, AMH, RHOX5 and CDKN1B expression was decreased while GATA4 expression was increased. Conclusions: In summary, we showed that feeding with crude garlic inhibited Leydig steroidogenic enzyme expression and Sertoli cell markers. These alterations might induce apoptosis in testicular germ cells.

Increases in norepinephrine release and ovarian cyst formation during ageing in the rat

Eric Acuna — 2009-06-16T00:00:00Z

Background: Depletion of ovarian follicles is associated with the end of reproductive function in ageing females. Recently, it has been described that this process parallels increases in the concentration of norepinephrine (NE) in the rat ovary. In sexually mature rats, experimentally-induced increases in the sympathetic tone of the ovary is causally related to ovarian cyst formation and deranged follicular development. Thus, there is a possibility that increased ovarian NE concentrations represent changes in the activity of sympathetic nerves, which consequently participate in the process of ovarian cyst formation observed during ageing in the human and experimental animal models. Methods: Sprague-Dawley rats between 6 and 14 months old were used to analyse the capacity of the ovary to release 3H-NE recently incorporated under transmural depolarisation in relation to changes in the ovarian follicular population. Morphometric analysis of ovarian follicles and real time PCR for Bcl2 and Bax mRNA were used to assess follicular atresia. Results: From 8 months old, the induced release of recently incorporated 3H-norepinephrine (3H-NE) from the ovary and ovarian NE concentrations increased, reaching their peak values at 12 months old and remained elevated up to 14 months old. Increases in sympathetic nerve activity paralleled changes in the follicular population, as well as disappearance of the corpus luteum. In contrast, luteinised follicles, precystic follicles, and cystic follicles increased. During this period, the relationship between Bax and Bcl2 mRNAs (the proapoptotic/antiapoptotic signals) increased, suggesting atresia as the principal mechanism contributing to the decreased follicular population. When NE tone was increased, the mRNA ratio favoured Bcl2 to Bax and antiapoptotic signals dominated this period of development. Thus, these changing ratios could be responsible for the increase in luteinised follicles, as well as precystic and cystic follicles. Conclusion: These data suggest that the ageing process in the ovary of the Sprague-Dawley rat is accompanied by an increased sympathetic tone of the ovary. Consequently, this sympathetic change could be related to a neuroendocrine-driven formation of a polycystic condition similar to that observed in the sympathetic-activated adult ovary.

NTPDases in the neuroendocrine hypothalamus: possible energy regulators of the positive gonadotrophin feedback

Attila Zsarnovszky — 2009-06-16T00:00:00Z

Background: Brain-derived ectonucleoside triphosphate diphosphohydrolases (NTPDases) have been known as plasma membrane-incorporated enzymes with their ATP-hydrolyzing domain outside of the cell. As such, these enzymes are thought to regulate purinergic intercellular signaling by hydrolyzing ATP to ADP-AMP, thus regulating the availability of specific ligands for various P2X and P2Y purinergic receptors. The role of NTPDases in the central nervous system is little understood. The two major reasons are the insufficient knowledge of the precise localization of these enzymes in neural structures, and the lack of specific inhibitors for the various NTPDases. To fill these gaps, we recently studied the presence of neuron-specific NTPDase3 in the mitochondria of hypothalamic excitatory neurons by morphological and functional methods. Results from those studies suggested that intramitochondrial regulation of ATP levels may play a permissive role in the neural regulation of physiological functions by tuning the level of ATP-carried energy that is needed for neuronal functions, such as neurotransmission and/or intracellular signaling.Presentation of the hypothesisIn the lack of highly specific inhibitors, the determination of the precise function and role of NTPDases is hardly feasable. Yet, here we attempt to find an approach to investigate a possible role for hypothalamic NTPDase3 in the initiation of the midcycle luteinizing hormone (LH) surge, as such a biological role was implied by our recent findings. Here we hypothesize that NTPDase-activity in neurons of the AN may play a permissive role in the regulation of the estrogen-induced pituitary LH-surge.Testing the hypothesisWe propose to test our hypothesis on ovariectomized rats, by stereotaxically injecting 17beta-estradiol and/or an NTPDase-inhibitor into the arcuate nucleus and determine the consequential levels of blood LH, mitochondrial respiration rates from arcuate nucleus synaptosomal preparations, NTPDase3-expression from arcuate nucleus tissue samples, all compared to sham and intact controls.Implications of the hypothesisResults from these studies may lead to the conclusion that estrogen may modulate the activity of mitochondrial, synapse-linked NTPDase3, and may show a correlation between mitochondrial NTPDase3-activity and the regulation of LH-release by estrogen.

Racial disparity in pathophysiologic pathways of preterm birth based on genetic variants

Ramkumar Menon — 2009-06-15T00:00:00Z

ObjectiveTo study pathophysiologic pathways in spontaneous preterm birth and possibly the racial disparity associating with maternal and fetal genetic variations, using bioinformatics tools. Methods: A large scale candidate gene association study was performed on 1442 SNPs in 130 genes in a case (preterm birth <36 weeks) control study (term birth >37 weeks). Both maternal and fetal DNA from Caucasians (172 cases and 198 controls) and 279 African-Americans (82 cases and 197 controls) were used. A single locus association (genotypic) analysis followed by hierarchical clustering was performed, where clustering was based on p values for significant associations within each race. Using Ingenuity Pathway Analysis (IPA) software, known pathophysiologic pathways in both races were determined. Results: From all SNPs entered into the analysis, the IPA mapped genes to specific disease functions. Gene variants in Caucasians were implicated in disease functions shared with other known disorders; specifically, dermatopathy, inflammation, and hematological disorders. This may reflect abnormal cervical ripening and decidual hemorrhage. In African-Americans inflammatory pathways were the most prevalent. In Caucasians, maternal gene variants showed the most prominent role in disease functions, whereas in African Americans it was fetal variants. The IPA software was used to generate molecular interaction maps that differed between races and also between maternal and fetal genetic variants. Conclusion: Differences at the genetic level revealed distinct disease functions and operational pathways in African Americans and Caucasians in spontaneous preterm birth. Differences in maternal and fetal contributions in pregnancy outcome are also different between African Americans and Caucasians. These results present a set of explicit testable hypotheses regarding genetic associations with preterm birth in African Americans and Caucasians

The impact of cocaine and heroin on the placental transfer of methadone

Antoine Malek — 2009-06-11T00:00:00Z

Background: Methadone is the therapeutic agent of choice for the treatment of opiate addiction in pregnancy. The co-consumption (heroin, cocaine) which may influence the effects of methadone is frequent. Therefore, the impact of cocaine and heroin on the placental transfer of methadone and the placental tissue was investigated under in vitro conditions. Methods: Placentae (n = 24) were ex-vivo perfused with medium (m) (control, n = 6), m plus methadone (n = 6), m plus methadone and cocaine (n = 6) or m plus methadone and heroin (n = 6). Placental functionality parameters like antipyrine permeability, glucose consumption, lactate production, hormone production (hCG and leptin), microparticles release and the expression of P-glycoprotein were analysed. Results: Methadone accumulated in placental tissue. Methadone alone decreased the transfer of antipyrine from 0.60 +/- 0.07 to 0.50 +/- 0.06 (fetal/maternal ratio, mean +/- SD, P < 0.01), whereas the combination with cocaine or heroin increased it (0.56 +/- 0.08 to 0.68 +/- 0.13, P = 0.03 and 0.58 +/- 0.21 to 0.71 +/- 0.24; P = 0.18). Microparticles (MPs) released from syncytiotrophoblast into maternal circuit increased by 30% after cocaine or heroin (P < 0.05) and the expression of P-glycoprotein in the tissue increased by ≥ 49% after any drug (P < 0.05). All other measured parameters did not show any significant effect when methadone was combined with cocaine or heroine. Conclusion: The combination of cocaine or heroin with methadone increase antipyrine permeability. Changes of MPs resemble findings seen in oxidative stress of syncytiotrophoblast.

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

Renato Salgado — 2009-06-05T00:00:00Z

Background: Remodeling 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. Methods: Uteri 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. Results: In 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. Conclusion: These 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.

Epigenetic regulation in mammalian preimplantation embryo development

Lingjun Shi — 2009-06-05T00:00:00Z

Preimplantation embryo development involves four stages: fertilization, cell cleavage, morula and blastocyst formation. During these stages, maternal and zygotic epigenetic factors play crucial roles. The gene expression profile is changed dramatically, chromatin is modified and core histone elements undergo significant changes. Each preimplantation embryo stage has its own characteristic epigenetic profile, consistent with the acquisition of the capacity to support development. Moreover, histone modifications such as methylation and acetylation as well as other epigenetic events can act as regulatory switches of gene transcription. Because the epigenetic profile is largely related to differentiation, epigenetic dysfunction can give rise to developmental abnormalities. Thus, epigenetic profiling of the embryo is of pivotal importance clinically. Given the importance of these aspects, this review will mainly focus on the epigenetic profile during preimplantation embryo development, as well as interactions between epigenetic and genetic regulation in these early developmental stages.