Ovulation in the tree shrew (Tupaia belangeri) induced by gonadotrophins

The induction of ovulation by exogenous gonadotrophins is an important approach for recovering oocytes used for studies on the reproductive biology of some mammals. In the present study, pregnant mare serum gonadotrophin (PMSG) and human chorionic gonadot

黄鳝cGnRH-Ⅱ的cDNA克隆与序列分析

促性腺激素释放激素(Gonadotropin-releasing hormone,GnRH)是一个保守的十肽神经家族激素,在脊椎动物的性腺发育和繁殖功能的维持方面起着重要的调控作用。本文通过运用RACE和RT-PCR方法,从黄鳝脑组织中克隆得到cGnRH-ⅡcDNA全序列,其核苷酸序列长度为617bp。该cDNA编码的cGnRH-Ⅱ的前体氨基酸序列结构组成与其他物种的cGnRH-Ⅱ前体结构一致,其推导的蛋白前体长度为83个氨基酸,包括一个信号肽、cGnRH-Ⅱ十肽和一个由蛋白水解位点(Gly-Lys-Ar

Molecular characterization of Chinese sturgeon gonadotropins and cellular distribution in pituitaries of mature and immature individuals

Chinese sturgeon (Acipenser sinensis) is a rare and endangered species, and also an important resource for the sturgeon aquaculture industry. To understand molecular characterization of Chinese sturgeon gonadotropins (GTHs), we cloned the full-length cDNAs of gonadotropin subunits common alpha (GTH-alpha), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from a pituitary cDNA library of mature female. Two subtypes of GTH-alpha were identified. The nucleotide sequences of A. sinensis common alpha I (AsGTH-alpha I), common alpha II (AsGTH-alpha II), FSH beta (AsFSH beta) and LH beta (AsLH beta) subunit cDNAs are 345, 363, 387 and 414 bp in length, and encode mature peptides of 115, 121, 129 and 138 aa, respectively. Then, three polyclonal antibodies were prepared from the in vitro expressed AsGTH-alpha I, AsFSH beta and AsLH beta mature proteins, respectively. Significant expression differences were revealed between immature and mature sturgeon pituitaries. Western blot detection and immunofluoresence localization revealed the existence of three-gonadotropin subunits (AsGTH-alpha, AsFSH beta and AsLH beta) in mature sturgeon pituitaries, but only AsFSH beta was detected in immature individual pituitaries during early stages in the sturgeon life, and obvious difference was observed between males and females. In males, AsFSH beta was expressed in 4-year-old individuals, whereas in females, AsFSH beta was just expressed in 5-year-old individuals. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

A perspective on fish gonad manipulation for biotechnical applications

The gonad is an essential organ for generating sperm and ova in vertebrates. This review describes several pilot studies on gonad gene manipulation and development in fish. With antisense RNA techniques, we suppressed the gonad development, and thus the fertility, of an antisense gonadotropin-releasing hormone (sGnRH) transgenic common carp. Then, using a tissue-specific exogenous gene excision strategy with sexual compensation, we knocked out the gonad-specific transgene. Under the control of the rainbow trout protamine promoter, the transgenic fish expressed the reporter gene eGFP specifically in the spermary. These results indicate that the fish gonad is a new model organ that can improve contemporary biotechnology experiments. Herein we discuss the potential of fish gonad manipulation for resolving important biosafety problems regarding transgenic fish generation and producing the new transgenic animal bioreactor.

Cloning and expression analysis in mature individuals of two chicken type-II GnRH (cGnRH-II) genes in common carp (Cyprinus carpio)

Gonadotropin-releasing hormone (GnRH) is a conservative neurodecapeptide family, which plays a crucial role in regulating the gonad development and in controlling the final sexual maturation in vertebrate. Two differing cGnRH-II cDNAs of common carp, namely cGnRH-II cDNA1 and cDNA2, were firstly cloned from the brain by rapid amplification of cDNA end (RACE) and reverse transcription- polymerase chain reaction (RT-PCR). The length of cGnRH-II cDNA1 and cDNA2 was 622 and 578 base pairs (bp), respectively. The cGnRH-II precursors encoded by two cDNAs consisted of 86 amino acids, including a signal peptide, cGnRH-II decapeptide and a GnRH-associated peptide (GAP) linked by a Gly-Lys-Arg proteolytic site. The results of intron trapping and Southern blot showed that two differing cGnRH-II genes in common carp genome were further identified, and that two genes might exist as a single copy. The multi-gene coding of common carp cGnRH-II gene offered novel evidence for gene duplication hypothesis. Using semi-quantitative RT-PCR, expression and relative expression levels of cGnRH-II genes were detected in five dissected brain regions, pituitary and gonad of common carp. With the exception of no mRNA2 in ovary, two cGnRH-II genes could be expressed in all the detected tissues. However, expression levels showed an apparent difference in different brain regions, pituitary and gonad. According to the expression characterization of cGnRH-II genes in brain areas, it was presumed that cGnRH-II might mainly work as the neurotransmitter and neuromodulator and also operate in the regulation for the GnRH releasing. Then, the expression of cGnRH-II genes in pituitary and gonad suggested that cGnRH-II might act as the autocrine or paracrine regulator.

Molecular analysis of silver crucian carp (Carassius auratus gibelio Bloch) clones by SCAR markers

Random amplified polymorphic DNA (RAPD) molecular markers specific for one, two or three clones have been identified from five gynogenetic clones of silver crucian carp (Carassius auratus gibelio Bloch) using RAPD markers developed earlier. In this study, three RAPD markers (RA1-PA, RA2-EF and RA4-D) produced by Opj-1, and two RAPD DNA fragments (RA3-PAD and RA5-D) produced by Opj-7, were selected for molecular cloning and sequencing. Sequence data indicated that there were identical 801-bp nucleotide sequences in the shared marker RA1-PA cloned respectively from clones P and A, and the shared marker RA2-EF (which was cloned from clones E and F), were also of identical 958-by nucleotide sequences. The nucleotide sequences of the shared marker RA3-PAD fragments were also similar for 1181 by among clones P, A and D. The specific fragment RA4-D was composed of 628 bp, and the fragment RA5-D from clone D contained 385 nucleotides. According to the nucleotide sequences, we designed and synthesized five pairs of sequence characterized amplified regions (SCAR) primers to identify the specific fragments in these gynogenetic clones of silver crucian carp. Only individuals from clones P and A amplified a specific band using a pair of SCI-PA primers synthesized according to the marker RA1-PA sequences, whereas no products were detected in individuals from clones D, E and F. The PCR products amplified using SC2-EF and SC3-PAD primers were as expected. Furthermore, the pair of SC4-D primers amplified specific bands only in individuals from clone D, although weak bands could be produced in all individuals of the five clones when lower annealing temperatures were used. However, an additional pair of SC5-D primers designed from the RA5-D marker sequences could amplify a DNA band in individuals from clones P, A and D, and the same weak band was produced in clone E, whereas no products were detected in individuals from clone F. Searches in GenBank revealed that the 385-bp DNA fragment from RA5-D was homologous to the 5' end of gonadotropin I beta subunit 2 gene and growth hormone gene. No homologous sequences were found for other markers in GenBank. The SCAR markers identified in this study will offer a powerful, easy, and rapid method for discrimination of different clones and for genetic analyses that examine their origins and unique reproductive modes in crucian carp. Furthermore, they will likely benefit future selective breeding programs as reliable and reproducible molecular markers. (C) 2001 Elsevier Science B.V. All rights reserved.

Estradiol stimulates growth hormone production in female goldfish

The effects of estradiol (E(2)) on growth hormone (GH) production was investigated in gonad-intact female goldfish. It was first necessary to generate a specific antibody for use in immunocytochemistry, Western, and dot-blot analyses of GH production. To accomplish this, grass carp GH (gcGH) cDNA was cloned by the reverse transcription polymerase chain reaction (RT-PCR) and expressed in Echerichia coli and a specific polyclonal antibody to recombinant gcGH was generated in the rabbit. In Western blot, the anti-gcGH antibody specifically immunoreacted with recombinant gcGH, purified natural common carp GH, and with a single 21.5-kDa GH form from pituitary extracts of grass carp, common carp, goldfish, and zebrafish but not salmon, trout, or tilapia. Intraperitoneal injection of the recombinant gcGH enhanced the growth rates of juvenile common carp demonstrating biological activity of this GH preparation. Electron microscopic studies showed that the anti-gcGH-I antibody specifically reacted with GH localized in the secretory granules of the goldfish somatotroph. Using anti-gcGH-I in a dot-blot assay, it was found that in vivo implantation of solid silastic pellets containing E(2) (100 mu g/g body weight for 5 days) increased pituitary GH content by 150% in female goldfish. In a second, independent study employing a previously characterized anticommon carp GH antibody for radioimmunoassay, it was found that E(2) increased pituitary GH content by 170% and serum GH levels by approximately 350%. The E(2)-induced hypersecretion of GH and increase in pituitary GH levels was not associated with changes in steady-state pituitary GH mRNA levels, suggesting that this sex steroid may enhance GH synthesis at the posttranscriptional or translational level. Previous observations indicate that GH can stimulate ovarian E(2) production. The present results show that E(2) can in turn stimulate GH production, indicating the existence of a novel pituitary GH-ovarian feedback system in goldfish. (C) 1997 Academic Press.

Alleviation of pre-exposure of mouse brain with low-dose C-12(6+) ion or Co-60 gamma-ray on male reproductive endocrine damages induced by subsequent high-dose irradiation

Irradiation has been widely reported to damage organisms by attacking on proteins, nucleic acid and lipids in cells. However, radiation hormesis after low-dose irradiation has become the focus of research in radiobiology in recent years. To investigate the effects of pre-exposure of mouse brain with low-dose C-12(6+) ion or Co-60 gamma (gamma)-ray on male reproductive endocrine capacity induced by subsequent high-dose irradiation, the brains of the B6C3F(1) hybrid strain male mice were irradiated with 0.05 Gy of C-12(6+) ion or Co-60 gamma-ray as the pre-exposure dose, and were then irradiated with 2 Gy as challenging irradiation dose at 4 h after pre-exposure. Serum pituitary gonadotropin hormones, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), testosterone, testis weight, sperm count and shape were measured on the 35th day after irradiation. The results showed that there was a significant reduction in the levels of serum FSH, LH, testosterone, testis weight and sperm count, and a significant increase in sperm abnormalities by irradiation of the mouse brain with 2 Gy of C-12(6+) ion or Co-60 gamma-ray. Moreover, the effects were more obvious in the group irradiated by C-12(6+) ion than in that irradiated by Co-60 gamma-ray. Pre-exposure with low-dose C-12(6+) ion or Co-60 gamma-ray significantly alleviated the harmful effects induced by a subsequent high-dose irradiation.

Rapid behavioral and genomic responses to social opportunity.

From primates to bees, social status regulates reproduction. In the cichlid fish Astatotilapia (Haplochromis) burtoni, subordinate males have reduced fertility and must become dominant to reproduce. This increase in sexual capacity is orchestrated by neurons in the preoptic area, which enlarge in response to dominance and increase expression of gonadotropin-releasing hormone 1 (GnRH1), a peptide critical for reproduction. Using a novel behavioral paradigm, we show for the first time that subordinate males can become dominant within minutes of an opportunity to do so, displaying dramatic changes in body coloration and behavior. We also found that social opportunity induced expression of the immediate-early gene egr-1 in the anterior preoptic area, peaking in regions with high densities of GnRH1 neurons, and not in brain regions that express the related peptides GnRH2 and GnRH3. This genomic response did not occur in stable subordinate or stable dominant males even though stable dominants, like ascending males, displayed dominance behaviors. Moreover, egr-1 in the optic tectum and the cerebellum was similarly induced in all experimental groups, showing that egr-1 induction in the anterior preoptic area of ascending males was specific to this brain region. Because egr-1 codes for a transcription factor important in neural plasticity, induction of egr-1 in the anterior preoptic area by social opportunity could be an early trigger in the molecular cascade that culminates in enhanced fertility and other long-term physiological changes associated with dominance.

Analysis of parameters of haemostasis in pre-eclamptic, normotensive pregnant and non-pregnant women.

Pregnancy is characterized by a state of heightened coagulation, which is exacerbated in pathological conditions such as pre-eclampsia (PET). PET is further associated with abnormal maternal inflammation and increased circulating microparticles (MP); however, a mechanistic link between these pathological features has never been established. It is proposed in this thesis that abnormal maternal inflammation is causally linked to pro-coagulant trophoblast MP shedding via a mechanism mediated by the pro-inflammatory cytokine tumour necrosis factor alpha (TNF), thereby contributing to maternal coagulopathies associated with PET. Using thromboelastography (TEG) and standard laboratory tests, haemostatic function was evaluated in PET and normotensive subjects at delivery and post-partum. Furthermore, the effects of the menstrual cycle and oral contraceptive (OC) use on haemostatic function were assessed in non-pregnant subjects in order to understand their influence on post-partum haemostasis. Plasma TNF and pro-coagulant MP levels were evaluated in the pregnant subjects. Using chorionic villi explants from human term placentas, MPs were quantified after TNF administration. The pro-coagulant potential of placental MPs was evaluated by TEG by spiking whole-blood with medium containing MPs from chorionic villi. TEG identified increased whole-blood coagulability in PET subjects at delivery, demonstrating its increased sensitivity over standard laboratory tests at identifying haemostatic alterations associated with PET. Haemostatic alterations were normalized by six weeks post-partum. TEG also identified cyclic haemostatic variations associated with OC use. Chorionic villi treated with TNF (1 ng/ml) shed significantly more MPs than untreated placentas. MPs from chorionic villi increased the coagulability of whole-blood. Together, results provide evidence supporting the concept that abnormal maternal inflammation is causally linked to the development of maternal coagulopathies in pregnancy complications. Moreover, TEG may be superior to standard laboratory tests in evaluating haemostasis in pregnant and non-pregnant subjects.

Pubertal impairment in Nhlh2

Pubertal development is impaired in mice lacking the basic helix-loop-helix transcription factor Nhlh2. The mechanisms underlying changes in reproduction in Nhlh2-deficient mice (Nhlh2-/-) are unclear. Here we show that hypothalamic gonadotropin-releasing hormone-1 (GnRH-1) content is reduced in adult Nhlh2-/- mice as is the number of GnRH-1 neurons localized to mid- and caudal hypothalamic regions. This reduction was detected postnatally after normal migration of GnRH-1 neurons within nasal regions had occurred. Phenotype rescue experiments showed that female Nhlh2-/- mice were responsive to estrogen treatment. In contrast, puberty could not be primed in female Nhlh2-/- mice with a GnRH-1 regimen. The adenohypophysis of Nhlh2-/- mice was hypoplastic although it contained a full complement of the five anterior pituitary cell types. GnRH-1 receptors (GnRHRs) were reduced in Nhlh2-/- pituitary gonadotropes as compared to wild type. In vitro assays indicated that Nhlh2 expression is regulated in parallel with GnRHR expression. However, direct transcriptional activity of Nhlh2 on the GnRHR promoter was not found. These results indicate that Nhlh2 plays a role in the development and functional maintenance of the hypothalamic-pituitarygonadal axis at least at two levels: 1) in the hypothalamus by regulating the number and distribution of GnRH-1 neurons and, 2) in the developing and mature adenohypophysis.

IMMUNOCYTOCHEMICAL DEMONSTRATION OF NEUROPEPTIDES IN THE CENTRAL-NERVOUS-SYSTEM OF THE ROUNDWORM, ASCARIS-SUUM (NEMATODA, ASCAROIDEA)

The localization and distribution of neuropeptides in the central nervous system of the pig roundworm, Ascaris suum, have been determined by an indirect immunofluorescence technique in conjunction with confocal microscopy. Antisera to 25 vertebrate peptides and two invertebrate peptides were used to screen the worm for immunoreactivity (IR). Immunostaining was obtained with antisera to pancreatic polypeptide (PP), peptide YY (PYY), neuropeptide Y (NPY), gastrin, cholecystokinin (CCK), substance P (SP), atrial natriuretic peptide (ANP), salmon gonadotropin-releasing hormone (SGnRH), mammalian gonadotropin-releasing hormone (MGnRH), chromogranin A (CGA) and FMRFamide. The most extensive patterns of IR occurred with antisera to PYY, FMRFamide and gastrin. IR was evident in nerve cells and fibres in the ganglia associated with the anterior nerve ring and in the main nerve cords and their commissures; IR to FMRFamide also occurred in the posterior nerve ring. Immunostaining for the other peptides was confined to the nerve cords, with the number of immunoreactive nerve fibres varying from peptide to peptide.

Potential biomarkers on gametogenesis process and in sperm quality of european EEL (Anguilla anguilla)

Dissertação de mestrado, Aquacultura, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015

Serum estradiol: oocyte ratio as a predictor of reproductive outcome: an analysis of data from >9000 IVF cycles in the Republic of Ireland

Purpose The purpose of this study was to evaluate the serum estradiol (E2) per oocyte ratio (EOR) as a function of selected embryology events and reproductive outcomes with IVF. Methods This retrospective analysis included all IVF cycles where oocyte collection and fresh transfer occurred between January 2001 and November 2012 at a single institution. Patients were divided by three age groups (<35, 35–39, and ≥40 years) and further stratified into nine groups based on EOR (measured in pmol/L/oocyte). Terminal serum E2 (pmol/mL) was recorded on day of hCG trigger administration, and fertilization rate, cleavage rate, number of good quality embryos, and reproductive outcomes were recorded for each IVF cycle. Results During the study interval, 9109 oocyte retrievals were performed for 5499 IVF patients (mean = 1.7 cycles/patient). A total of 63.4 % of transfers were performed on day 3 (n = 4926), while 36.6 % were carried out on day 5 (n = 2843). Clinical pregnancy rates were highest in patients with EOR of 250–750 and declined as this ratio increased, independent of patient age. While the odds ratio (OR) for clinical pregnancy where EOR = 250–750 vs. EOR > 1500 was 3.4 (p < 0.001; 95 % CI 2.67–4.34), no statistically significant correlation was seen in fertilization, cleavage rates or number of good quality embryos as a function of EOR. Conclusions Predicting reproductive outcomes with IVF has great utility both for patients and providers. The former have the opportunity to build realistic expectations, and the latter are better able to counsel according to measured clinical parameters. A better understanding of follicular dynamics and ovarian response to gonadotropin stimulation could optimize IVF treatments going forward.

Molecular characterization and phenotypic expression of mutations in genes for gonadotropins and their receptors in humans.

Gonadotropin hormones undergo important dynamic changes during life. Their rise during puberty stimulates gonadal steroid secretion, triggering the development of secondary sexual characteristics and the acquisition of fertility. The full spectrum of possible mutations and polymorphisms in the human gonadotropins and in their receptor genes has been described in recent years. Patients harboring these mutations display a very wide range of phenotypes affecting all aspects of the reproductive axis. An important insight provided by the careful study of these patients lies in the striking gender differences in the phenotypes associated with a given mutation. As a result, the careful study of these rare patients has allowed us to better define the respective roles of luteinizing hormone and follicle-stimulating hormone in normal human pubertal development and in the achievement of full fertility potential in either males or females. In this work, we describe briefly the known mutations in the genes for both gonadotropins and their receptors, and discuss their genotype/phenotype correlations in light of these important gender differences.