Molecular insights into the causes of male infertility

Infertility is a reproductive health problem that affects many couples in the human population. About 13–18% of couple suffers from it and approximately one-half of all cases can be traced to either partner. Regardless of whether it is primary or secondary infertility, affected couples suffer from enormous emotional and psychological trauma and it can constitute a major life crisis in the social context. Many cases of idiopathic infertility have a genetic or molecular basis. The knowledge of the molecular genetics of male infertility is developing rapidly, new spermatogenic genes are being discovered and molecular diagnostic approaches (DNA chips) established. This will immensely help diagnostic and therapeutic approaches to alleviate human infertility. The present review provides an overview of the causes of human infertility, particularly the molecular basis of male infertility and its implications for clinical practice

Directed differentiation of mouse P19 embryonal carcinoma cells to neural cells in a serum- and retinoic acid-free culture medium

P19 embryonal carcinoma cells (EC-cells) provide a simple and robust culture system for studying neural development. Most protocols developed so far for directing neural differentiation of P19 cells depend on the use of culture medium supplemented with retinoic acid (RA) and serum, which has an undefined composition. Hence, such protocols are not suitable for many molecular studies. In this study, we achieved neural differentiation of P19 cells in a serum- and RA-free culture medium by employing the knockout serum replacement (KSR) supplement. In the KSR-containing medium, P19 cells underwent predominant differentiation into neural lineage and by day 12 of culture, neural cells were present in 100% of P19-derived embryoid bodies (EBs). This was consistently accompanied by the increased expression of various neural lineage-associated markers during the course of differentiation. P19-derived neural cells comprised of NES+ neural progenitors (degrees similar to 46%), TUBB3(+) immature neurons (similar to 6%), MAP2(+) mature neurons (similar to 2%), and GFAP(+) astrocytes (similar to 50%). A heterogeneous neuronal population consisting of glutamatergic, GABAergic, serotonergic, and dopaminergic neurons was generated. Taken together, our study shows that the KSR medium is suitable for the differentiation of P19 cells to neural lineage without requiring additional (serum and RA) supplements. This stem cell differentiation system could be utilized for gaining mechanistic insights into neural differentiation and for identifying potential neuroactive compounds

Identification and molecular characterisation of a biotin-binding protein distinct from avidin of chicken egg white and comparison with yolk biotin-binding protein

By immunological and biochemical methods a biotin-binding protein, distinct from avidin, has been shown to be present in chicken egg white. This vitamin-binding protein (Mr 67 000) bound [14C]biotin, displayed thermally induced biotin exchange reaction and exhibited gross immunological cross-reactivity with the purified yolk biotin-binding protein. In vitro labelling of soluble proteins with radioactive amino acids in the oviduct tissue explants from estrogenised chicks revealed that approx. 2% of the total radioactive proteins was immunoprecipitated with anti-yolk biotin-binding protein antibodies. The protein could be purified to homogeneity by employing ion-exchange chromatography on DEAE-cellulose and biotin-AH Sepharose affinity chromatography. The purified protein specifically bound [14C]biotin, and exhibited complete immunological homology with the yolk biotin-binding protein but not with avidin. Its electrophoretic mobility (at pH 8.3), acidic nature, biotin-binding characteristics, immunological cross-reactivity and tryptic peptide maps were very similar to that of yolk biotin-binding protein, and not avidin

Pregnancy suppression in the bonnet monkey by active immunisation with chicken riboflavin carrier protein

Five healthy female bonnet monkeys (Macaca radiata) of proven fertility and normal menstrual cyclicity have been actively immunised with the purified chicken egg white riboflavin carrier protein (cRCP). All the immunised animals exhibited specific antibodies to cRCP and the immunopotencies of their sera varied from 200 to 840 μ g/ml at equivalent point. A definite fraction of antibodies in these sera specifically recognized the purified and 125I-labelled monkey RCP. Immunisation per se had no adverse effect on the animals' menstrual cyclicity, circulating levels of estrogen and progesterone and the riboflavin status as reflected by glutathione reductase activities and the total flavin contents of the erythrocytes. The fertility of these animals was monitored for a period extending up to 3 years after primary immunisation. Four out of the five animals exhibited termination of pregnancy once or more than once depending on their antibody titers. Towards the end of the study period, when the immune response was poor, all the animals delivered normal babies at term. Circulating anti-cRCP antibodies were monitored by 125I-labelled cRCP binding. The results show that pregnancy termination, owing to immuno-neutralisation of monkey RCP, occurred only in animals which had sufficiently high antibody titers. If the titers fell below a critical threshold level the pregnancies were carried to term

Ascorbic acid-mediated enhanced cardiomyocyte differentiation of mouse ES-cells involves interplay of DNA methylation and multiple-signals

Embryonic stem cells (ES-cells) provide a good model system to study lineage-specific differentiation. Though, the differentiation of ES-cells to cardiomyocytes is documented, a clear understanding of the molecular mechanism of differentiation and improved functional-differentiation efficiency are yet to be achieved. In this regard, ascorbic acid (Aa) is shown to be one of the effective cardiac inducers in ES-cells. But, its mechanism is poorly understood. We therefore, investigated the mechanism of Aa-mediated cardiomyocyte differentiation of ES-cells. Here, we describe the potential involvement of epigenetic (DNA methylation) as well as integrin-and Erk-signaling systems during cardiomyocyte differentiation. Transgenic GS-2 ES-cells and wild-type D3 ES-cells were differentiated to cardiomyocytes, in the presence or absence of Aa and with or without inhibitors of Erk-, collagen-and integrin-pathways. At specific time points, differentiated states of ES-cells were scored by gene expression analyses and the proportion of functional cTnI(+) cardiomyocytes. DNA methylation changes of Isl-1, BMP-2, GATA-4 and alpha-MHC in cardiogenic cells, following stimulation with Aa, were analyzed by using methylation specific PCR (MSP). We observed that Aa, when applied in initial phase of ES-cell differentiation, consistently enhanced cardiac differentiation (99%) over that observed during spontaneous differentiation (70%). This was associated with enhanced expressions of cardiogenesis-associated genes. A two-fold increase in cTnI(+) cells was observed, with appropriate myofibril arrangement. The observed effect of Aa was due to enhanced collagen and integrin signaling, coupled with a high p-ERK1/2 expression, downstream. Besides, the involvement of DNA methylation in regulating the expression of cardiac genes i.e., Isl-1 and a-MHC was also observed. Overall, this study, for the first time, demonstrates that Aa-mediated cardiac enhancement is brought about, mechanistically, through the interplay of epigenetic changes in DNA methylation of cardiac genes (Isl-1 and a-MHC) and integrin signaling system

Isolation and characterisation of a biotin-binding protein from the pregnant-rat serum and comparison with that from the chicken egg-yolk

A biotin-binding protein exhibiting partial immunological cross-reactivity with the purified chicken egg-yolk biotin-binding protein has been detected, for the first time, in the sera of pregnant/estrogenised female rats but not of the normal males. This protein, purified by affinity chromatography on a biotin-AH-Sepharose was homogeneous by electrophoretic and immunological criteria. It was a glycoprotein of Mr 66 000 without any detectable subunites, had a pI 4.1, and specifically bound [14C]biotin. Several structural and functional features of the biotin-binding protein of rat and chicken were found to be similar. These included immunological cross-reactivity, acidic and glycoprotein nature, ability to tightly bind [14C]biotin, estrogen stimulation for their appearance in circulation, and the pattern of distribution of radioiodinated peptides upon proteolysis with trypsin

Successful Development of Viable Blastocysts From Enhanced Green Fluorescent Protein Transgene-Microinjected Mouse Embryos:Comparison of Culture Media

To improve efficiency of transgenesis, we compared M16 and CZB embryo culture media, supporting development to blastocysts of FVB/N mouse pronuclear-eggs, microinjected with enhanced green fluorescent protein (EGFP) transgene. When EGFP-injected-eggs were cultured (120 hr), blastocyst development was significantly (P<0.03) higher in M16 medium (72.5 \pm 2.4%) than that in CZB (13.2 \pm 4.3%) or CZBG (CZB with 5.6 mM glucose at 48 hr culture) (62.1 \pm 3.7%) media. Blastocyst development of noninjected embryos was higher in M16 (92.0 \pm 2.6%) and CZBG (83.9 \pm 3.9%) media than in CZB (31.9 \pm 2.8%) medium (P<0.0001). However, percentages of morulae at 72 hr were comparable in all treatments. Developed blastocysts were better in M16 than in CZB or CZBG media. Consistent with this, mean cell number per blastocyst, developed from injected embryos, was significantly (P<0.002) higher in M16 medium (79.6), than those in CZB (31.3) or CZBG media (60.7); similar with noninjected embryos. Cell allocation to trophectoderm (TE) and inner cell mass (ICM), i.e., TE:ICM ratio, for injected blastocysts in M16 (3.0) was less than (P<0.05) those in CZB (4.2) and CZBG (4.4) media; similar with noninjected blastocysts. Moreover, blastocysts, developed in M16 and CZBG media, hatched, attached, and exhibited trophoblast outgrowth; 18% of them showed EGFP-expression. Importantly, blastocysts from M16 medium produced live transgenic green pups (11%) following embryo transfer. Taken together, our results indicate that supplementation of glucose, at 48 hr of culture (CZBG), is required for morula to blastocyst transition; M16 medium, containing glucose from the beginning of culture, is superior to CZB or CZBG for supporting development of biologically viable blastocysts from EGFPtransgene injected mouse embryos

Identification and molecular characterization of a biotin-binding protein distinct from avidin of chicken egg white and comparison with yolk biotin-binding protein

By immunol. and biochem. methods a biotin-binding protein, distinct from avidin, was shown to be present in chicken egg white. This vitamin-binding protein (mol. wt. 67,000) bound [14C]biotin, displayed thermally induced biotin exchange reaction, and exhibited gross immunol. cross-reactivity with the purified yolk biotin-binding protein. In vitro labeling of sol. proteins with radioactive amino acids in the oviduct tissue explants from estrogenized chicks revealed that .apprx.2% of the total radioactive proteins immunopptd. with anti-yolk biotin-binding protein antibodies. The protein was purified to homogeneity by ion-exchange chromatog. on DEAE-cellulose and biotin-AH Sepharose affinity chromatog. The purified protein specifically bound [14C]biotin, and exhibited complete immunol. homol. with the yolk biotin-binding protein but not with avidin. Its electrophoretic mobility (at pH 8.3), acidic nature, biotin-binding characteristics, immunol. cross-reactivity, and tryptic peptide maps were very similar to those of yolk biotin-binding protein, and not avidin

Pregnancy suppression in the bonnet monkey by active immunisation with chicken riboflavin carrier protein

Five healthy female bonnet monkeys (Macaca radiata) of proven fertility and normal menstrual cyclicity have been actively immunised with the purified chicken egg white riboflavin carrier protein (cRCP). All the immunised animals exhibited specific antibodies to cRCP and the immunopotencies of their sera varied at 200-840 \mu g/ml at equivalent point. A definite fraction of antibodies in these sera specifically recognized the purified and $^{125}I-labelled$ monkey RCP. Immunisation per se had no adverse effect on the animals menstrual cyclicity, circulating levels of estrogen and progesterone and the riboflavin status as reflected by glutathione reductase activities and the total flavin contents of the erythrocytes. The fertility of these animals was monitored for a period extending upto 3 years after primary immunisation. Four out of the 5 animals exhibited termination of pregnancy once or more than once depending on their antibody titers. Towards the end of the study period, when the immune response was poor, all the animals delivered normal babies at term. Circulating anti-cRCP antibodies were monitored by $^{125}I-labelled$ cRCP binding. The results show that pregnancy termination, owing to immuno-neutralization of monkey RCP, occurred only in animals which had sufficiently high antibody titers. If the titers fell below a critical threshold level the pregnancies were carried to term