Therapeutic effects of chitosan-embedded vitamin C, E nanoparticles against cisplatin-induced gametogenic and androgenic toxicity in adult male rats.

Cisplatin, an anticancer drug used in treating various types of cancers, can cause reproductive toxicities during chemotherapy. Keeping this in view, the present study was designed to investigate the possible protective effects of normal vitamin C and E and vitamin C and E nanoparticles (embedded in chitosan) against cisplatin-induced reproductive toxicities. Vitamins C, E, and their nanoparticles in this regard proved to be an effective therapy. The work aimed to treat cisplatin-induced reproductive toxicities through vitamin C and E and their nanoparticles. Cisplatin exposure caused significant reduction in the weight, testosterone level, and changed lipid profile. Similarly, cisplatin induced significant widespread testicular atrophy and testicular lesions as evidenced by the gaps in the epithelium and loss of differentiating germ cells. Vitamin C and E and their nanoparticles rescued the weight, testosterone level, and testicular disturbances, which is associated with improved histological view of testicular tissues. The current study highlights evidence that designing a medication of vitamin C and E nanoparticles is useful in mitigating cisplatin-induced reproductive toxicity in cancerous male patients underlying chemotherapy.

SIRT1 Expression and Regulation in the Primate Testis.

The epigenetic mechanisms controlling germ cell development and differentiation are still not well understood. Sirtuin-1 (SIRT1) is a nicotinamide adenosine dinucleotide (NAD)-dependent histone deacetylase and belongs to the sirtuin family of deacetylases. It catalyzes the removal of acetyl groups from a number of protein substrates. Some studies reported a role of SIRT1 in the central and peripheral regulation of reproduction in various non-primate species. However, testicular SIRT1 expression and its possible role in the testis have not been analyzed in primates. Here, we document expression of SIRT1 in testes of different primates and some non-primate species. SIRT1 is expressed mainly in the cells of seminiferous tubules, particularly in germ cells. The majority of SIRT1-positive germ cells were in the meiotic and postmeiotic phase of differentiation. However, SIRT1 expression was also observed in selected premeiotic germ cells, i.e., spermatogonia. SIRT1 co-localized in spermatogonia with irisin, an endocrine factor specifically expressed in primate spermatogonia. In marmoset testicular explant cultures, SIRT1 transcript levels are upregulated by the addition of irisin as compared to untreated controls explants. Rhesus macaques are seasonal breeders with high testicular activity in winter and low testicular activity in summer. Of note, SIRT1 mRNA and SIRT1 protein expression are changed between nonbreeding (low spermatogenesis) and breeding (high spermatogenesis) season. Our data suggest that SIRT1 is a relevant factor for the regulation of spermatogenesis in primates. Further mechanistic studies are required to better understand the role of SIRT1 during spermatogenesis.

In-silico Analyses of Disease Causing Mutations in SLURP1 Gene.

The SLURP1 (secreted LY6/urokinase type plasminogen activator receptor related protein-1) belongs to the gene family of urokinase, a type of plasminogen activator receptor (uPAR). Mutations in the SLURP1 have been reported to cause serious genetic problems of skin, Mal De Meleda, and malignancies. With the advancement of computational tools, it became possible to predict the potential impact of gene variants on the structure and function of protein. Therefore, in present study, we aimed to perform in-silico analyses of the disease causing SLURP1 mutations using online tools. In-total, 21 variants occurring in coding and non-coding regions of SLURP1 were found from public databases. In curated data, we have found 57.14% (12/21) missense, 23.81% (5/21) splice site, 9.52% (2/21) nonsense, 4.76% (1/21) deletion, and 4.76% (1/21) frameshift mutations. Moreover, heterogeneity in genotypes and phenotypes, along with 7 hotspot points in SLURP1 has been noted. In-silico analyses of the subjected variants have depicted a range of pathogenicity by combinatorial predictions of different tools from being lowly to highly pathogenic. Thus, the present study paves a platform to link computational analyses of mutations for important regulatory genes that can be undertaken for their phenotypes and their correlation with the disease status in case control studies.

Whole Exome Sequencing Revealed a Novel Nonsense Variant in the GNRHR Gene Causing Normosmic Hypogonadotropic Hypogonadism in a Pakistani Family.

BACKGROUND: Congenital hypogonadotropic hypogonadism (CHH) is a heterogeneous disorder characterized by delayed or loss of puberty and infertility due to functional deficiency in the hypothalamic gonadotropin-releasing hormone (GnRH). CHH can be classified into 2 subtypes on the basis of olfaction: Kallmann syndrome and normosmic CHH (nCHH). The spectrum of genetic variants causing CHH is continually expanding. Here, we recruited a consanguineous Pakistani family having 2 male and 2 female infertile patients diagnosed with idiopathic nCHH. AIMS: The aim of this study was to investigate the genetic cause of nCHH in the family. METHODS: Clinical and physical analyses were performed for the patients. Genetic analysis was carried out using whole exome and Sanger sequencing. RESULTS: Clinical and physical investigations confirmed low levels of gonadotropins and failure of secondary sexual development in the patients. Genetic analysis identified a novel nonsense mutation (chr4: g.68619942G>A, c.112C>T, p.Arg38*) in the gonadotropin-releasing hormone receptor gene (GNRHR) recessively co-segregating with nCHH in this family. All the patients are homozygous and their parents are heterozygous carriers, while normal siblings are heterozygous carriers or wild-type for this mutation, indicating that the identified mutation is pathogenic for nCHH in the family. CONCLUSION: We report the first homozygous nonsense mutation in the GNRHR gene (chr4: g. 68619942G>A, c.112C>T, p. Arg38*) that is associated with familial nCHH. Hence, our study displayed a good correlation of the genotype and phenotype of nCHH patients.

Irisin in the primate hypothalamus and its effect on GnRH in vitro.

Irisin, encoded by the FNDC5 gene, is a recently discovered endocrine factor mainly secreted as a myokine and adipokine. However, irisin/FNDC5 expression has also been reported in different other organs including components of the reproductive axis. Yet, there is the scarcity of data on FNDC5/irisin expression, regulation and its reproductive effects, particularly in primates. Here, we report the expression of FNDC5/irisin, along with PGC1A (peroxisome proliferator-activated receptor gamma coactivator 1-alpha) and ERRA (estrogen-related receptor alpha), in components of the reproductive axis of marmoset monkeys. Hypothalamic FNDC5 and ERRA transcript levels are developmentally regulated in both male and female. We further uncovered sex-specific differences in FNDC5, ERRA and PGC1A expression in muscle and the reproductive axis. Moreover, irisin and ERRα co-localize in the marmoset hypothalamus. Additionally, in the arcuate nucleus of rhesus monkeys, the number of irisin+ cells was significantly increased in short-term fasted monkeys as compared to ad libitum-fed monkeys. More importantly, we observed putative interaction of irisin-immunoreactive fibers and few GnRH-immunoreactive cell bodies in the mediobasal hypothalamus of the rhesus monkeys. Functionally, we noted a stimulatory effect of irisin on GnRH synthesis and release in mouse hypothalamic neuronal GT1-7 cells. In summary, our findings show that FNDC5 and irisin are developmentally, metabolic-status dependently and sex-specifically expressed in the primate hypothalamic-pituitary-gonadal axis and exert a stimulatory effect on GnRH expression and release in mouse hypothalamic cells. Further studies are required to confirm the reproductive effects of irisin in vivo and to illuminate the mechanisms of its regulation.

Dynamic Regulation of Hypothalamic DMXL2, KISS1, and RFRP Expression During Postnatal Development in Non-Human Primates.

The neurobiological mechanism of puberty onset in primates is currently only partly understood. A recent study reported an important role of Dmx-like 2 (DMXL2), a gene encoding rabconnectin-3α vesicular protein, in human subjects with mental retardation and neuroendocrine impairment of reproduction. To further characterize the potential role of DMXL2 in the regulation of reproduction, we analyzed the expression of DMXL2 in hypothalami of newborn, infantile, juvenile, pubertal, and postpubertal female and male common marmoset monkeys. Additionally, as the relative hypothalamic levels of gonadotropin-inhibitory hormone (GnIH) transcript during postnatal development are unknown in primates, we also quantified messenger RNA (mRNA) levels of RFRP, a gene encoding GnIH. Moreover, the transcript levels of kisspeptin, a well-known regulator of the hypothalamic neurohormonal axis controlling reproduction, were also checked. Transcript and protein levels of DMXL2 and Kiss1 transcript levels increase from the newborn to the infantile and from the juvenile (prepubertal) to the pubertal and the postpubertal period. We also noted a clear upsurge in RFRP transcript levels in the prepubertal period. In conclusion, the hypothalamic expressions of Kiss1 and DMXL2 mRNA increase during infantile, pubertal, and adult stages compared to newborn and juvenile stages in common marmoset monkeys. In contrast, the expression of RFRP mRNA upsurges in juvenile monkeys. Further mechanistic studies are needed to characterize the potential inhibitory role of the GnIH-GPR147 signaling in the prepubertal period and the role of DMXL2 in the molecular cascade regulating the neuroendocrine reproductive axis in primates.

Hypothesis: Irisin is a metabolic trigger for the activation of the neurohormonal axis governing puberty onset.

A large body of data suggests that body weight influences puberty onset and adult reproduction. However, the underlying mechanism of how body weight influences puberty onset and fertility is not completely understood. The hypothalamic neuronal circuit regulating reproduction is restrained by inhibitory signals during childhood. At the time of puberty, these inhibitory signals are weakened and supplanted by stimulatory signals that, in turn, stimulate the release of gonadotropin-releasing hormone (GnRH) - a hypothalamic neuropeptide governing reproduction. A number of studies, however, suggest that puberty commencement occurs when body (fat) weight reaches a certain threshold, which is critical for the initiation of puberty and for support of the adult reproductive function. Previously, various signals have been studied which might link body (fat) weight-related information to the hypothalamic neuronal network regulating reproduction. However, the nature of the signal(s) that may link body fat and/or muscle mass with the hypothalamic neuronal network governing reproduction is still unclear. It has been intuitively speculated that augmentation of such signal(s) will cause a restriction of inhibitory input and activation of stimulatory input to GnRH secreting neurons at the time of puberty onset. Therefore, the unveiling of such signal(s) will greatly help in understanding the mechanism of puberty onset. Recently, it has been shown that expression of fibronectin type III domain containing-5 (FNDC5) mRNA in central and peripheral tissues upsurges during postnatal development, especially around the time of puberty onset. Moreover, the systemic level of irisin - one of the protein products of the FNDC5 gene that is secreted as myokine and adipokine - also rises during postnatal development and correlates with the timing of puberty onset. Therefore, we propose here that irisin might serve as a possible signal for linking body fat/muscle mass with the hypothalamic center governing reproductive function. We hypothesize that irisin acts as a trigger for the activation of the hypothalamic neuronal network monitoring the onset of puberty.

Outcome of VVF repair without omental interposition.

OBJECTIVE: To find out the outcome in cases of vesicovaginal fistula repair. METHODS: The descriptive study was conducted at the District Headquarter Hospital, Timergara, Lower Dir, Pakistan, from November 1, 2011 to November 2013, and comprised all patients admitted in Urology unit with vesicovaginal fistula. Repair was done with either transabdominal or transvaginal approach. Repair technique involved good tissue separation, interrupted sutures, and no omental interposition. Follow-up was of three months. RESULTS: There were 30 patients available, but 2(6.6%) were excluded. Among the remaining 28(93.3%) patients dehiscence was not noted in any patient, while only 4(14.3%) patients developed mild urinary tract infection. There were no intraoperative or postoperative deaths. CONCLUSIONS: Transvaginal or Transabdominal repair of vesicovaginal fistula is successful treatment modality if good dissection and tissue separation is applied with interrupted suturing. Omental interposition is not essential for good healing.

Kisspeptin signalling in the physiology and pathophysiology of the urogenital system.

Kisspeptin is a peptide hormone, which signals via the G-protein-coupled kisspeptin receptor (KISS1R). Kisspeptin-KISS1R signalling has been implicated in various physiological and pathophysiological processes in the urogenital system, including critical roles in ovarian function as a key player in the regulation of oocyte development. Kisspeptin also has roles in several different functions of the male reproductive tract, such as spermatogenesis and sperm capacitation, and is also thought to be involved in kidney physiology - studies in preclinical animal models have reported that expression of kisspeptin and/or KISS1R is altered in chronically impaired kidneys. The wider importance of kisspeptin action in the urogenital tract has been highlighted by the finding that it suppresses metastasis of urogenital carcinomas; besides the possible therapeutic potential of this finding, tissue and tumour-stage-specific alterations in kisspeptin and KISS1/KISS1R expression could potentially be used as biomarkers for the diagnosis and prognosis of urogenital carcinomas.

Fasting induced kisspeptin signaling suppression is regulated by glutamate mediated cues in adult male rhesus macaque (Macaca mulatta).

Kisspeptin signaling is suppressed by short term fasting. It has been reported that hypothalamic Kiss1 and Kiss1r mRNA expression decreased after 48h of fasting in male rhesus monkey. But the mechanism involved in the reduction of kisspeptin signaling after 48h of fasting is unknown. Recent studies have suggested the role of afferent excitatory and inhibitory pathways in the regulation of kisspeptin neurons. Therefore, this study was designed to observe the changes in the glutamate and GABA signaling during fed and 48h fasting states by performing immunofluorescence to examine the interaction of kisspeptin neurons with NR1 subunit of NMDA receptors and by performing SYBR green qRT-PCR to measure and quantify the levels of Kiss1, Kiss1r, NR1 and GAD67 mRNA in the POA and MBH of adult male rhesus macaque (Macaca mulatta) during 48h of fasting (n=2) and fed ad libitum (n=2). Plasma testosterone (p<0.05) and blood glucose levels were significantly (p<0.001) decreased after short term fasting. Our results clearly showed that expression of hypothalamic Kiss1, Kiss1r and NR1 mRNA was significantly (p<0.05) reduced in adult male rhesus monkeys which were fasted for 48h as compared to those which were fed ad libitum. There was no clear difference in the GAD67 mRNA contents between the two groups. Number of kisspeptin neurons and the interactions of kisspeptin neurons with NR1 were significantly (p<0.05) reduced after 48h fasting. These observations suggest that decreased kisspeptin signaling during fasting may occur due to reduction in glutamatergic inputs to kisspeptin neurons. Our results also suggest that fasting induced suppression of kisspeptin signaling is not mediated through GABAergic neurons.

Insight into the serum kisspeptin levels in infertile males.

BACKGROUND: Regulation of reproduction is now considered to be carried out by the kisspeptin and its receptor, GPR54 or Kiss1r. Mutations of either Kiss1 or Kiss1r in humans and mice result in profound hypogonadotropic hypogonadism. The present study was aimed to determine whether the levels of kisspeptin are associated with male infertility. METHODOLOGY: The study involved 176 male subjects aged 18 - 50 years including 26 fertile and 150 infertile. Infertile subjects were further subdivided according to WHO guidelines of semen analysis into 22 asthenozoospermia, 08 asthenoteratozoospermia, 18 azoospermia, 58 normozoospermia, 06 oligozoospermia, 12 oligoasthenozoospermia and 26 oligoasthenoteratozoospermia. Thorough clinical examinations excluded those suffering from chronic health problems. Serum kisspeptin levels were measured by enzyme immunoassay (EIA) and follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone were estimated by chemiluminescence assay (CLIA). RESULTS: The results of the present study have revealed that kisspeptin levels were significantly lower in all infertile males as compared to the fertile males. Significantly low LH and testosterone levels were observed in all infertile groups as compared to fertile group. FSH levels were significantly lower in normozoospermic and azoospermic as compared to fertile males, while no significant difference was observed between the other infertile and fertile group. CONCLUSION: The study revealed that serum kisspeptin levels were observed significantly lower in the infertile as compared to fertile males, indicating that the kisspeptin might be associated with the fertility problems in males.

Kisspeptin as a link between metabolism and reproduction: evidences from rodent and primate studies.

Changes in metabolic status gate reproductive activity by still incompletely deciphered mechanisms. Many neuropeptides have been shown to be involved in restraining hypothalamic gonadotropin releasing hormone (GnRH) release under conditions of negative energy balance. Broadly, on the basis of their effect on feeding, these can be grouped as orexigenic and anorexigenic neuropeptides. Reciprocally correlated, in response to changes in systemic concentrations of metabolic hormones, the secretion of orexigenic neuropeptides increases while that of anorexigenic neuropeptides decreases during conditions of food restriction. Recently, kisspeptin signaling in hypothalamus has appeared as a pivotal regulator of the GnRH pulse generator. Kisspeptin apparently does not affect feeding, but in light of accumulating data, it has emerged as one of the major conduits in relaying body metabolic status information to GnRH neurons. The present review examines such data obtained from rodent and primate models, which suggest kisspeptin-Kiss1r signaling as a possible pathway providing a link between metabolism and reproduction.

GnRH-deficient phenotypes in humans and mice with heterozygous variants in KISS1/Kiss1.

CONTEXT: KISS1 is a candidate gene for GnRH deficiency. OBJECTIVE: Our objective was to identify deleterious mutations in KISS1. PATIENTS AND METHODS: DNA sequencing and assessment of the effects of rare sequence variants (RSV) were conducted in 1025 probands with GnRH-deficient conditions. RESULTS: Fifteen probands harbored 10 heterozygous RSV in KISS1 seen in less than 1% of control subjects. Of the variants that reside within the mature kisspeptin peptide, p.F117L (but not p.S77I, p.Q82K, p.H90D, or p.P110T) reduces inositol phosphate generation. Of the variants that lie within the coding region but outside the mature peptide, p.G35S and p.C53R (but not p.A129V) are predicted in silico to be deleterious. Of the variants that lie outside the coding region, one (g.1-3659C→T) impairs transcription in vitro, and another (c.1-7C→T) lies within the consensus Kozak sequence. Of five probands tested, four had abnormal baseline LH pulse patterns. In mice, testosterone decreases with heterozygous loss of Kiss1 and Kiss1r alleles (wild-type, 274 ± 99, to double heterozygotes, 69 ± 16 ng/dl; r(2) = 0.13; P = 0.03). Kiss1/Kiss1r double-heterozygote males have shorter anogenital distances (13.0 ± 0.2 vs. 15.6 ± 0.2 mm at P34, P < 0.001), females have longer estrous cycles (7.4 ± 0.2 vs. 5.6 ± 0.2 d, P < 0.01), and mating pairs have decreased litter frequency (0.59 ± 0.09 vs. 0.71 ± 0.06 litters/month, P < 0.04) and size (3.5 ± 0.2 vs. 5.4 ± 0.3 pups/litter, P < 0.001) compared with wild-type mice. CONCLUSIONS: Deleterious, heterozygous RSV in KISS1 exist at a low frequency in GnRH-deficient patients as well as in the general population in presumably normal individuals. As in Kiss1(+/-)/Kiss1r(+/-) mice, heterozygous KISS1 variants in humans may work with other genetic and/or environmental factors to cause abnormal reproductive function.

The kisspeptin signaling pathway and its role in human isolated GnRH deficiency.

Amplification of the neurosecretory activity of the GnRH system is the defining neuroendocrine event for sexual maturation. The physiological mechanisms that drive GnRH secretion at puberty have been difficult to identify but the discovery in 2003 that the G protein coupled receptor KISS1R is a key regulator of pubertal development in mice and men has ushered in a new chapter in reproductive neuroendocrinology. KISS1R is activated by endogenous peptides derived from a precursor protein, kisspeptin. Despite kisspeptin's importance in driving the reproductive cascade, relatively few patients with GnRH deficient states and mutations in the kisspeptin pathway have been described. Yet, these cases, coupled with loss-of-function mouse models, provide unique and complementary information into the biological role of this signaling system in the control of GnRH secretion. This article will examine some of the subtleties in genotype-phenotype correlations in both mice and men carrying disabling mutations in the kisspeptin pathway.

In hospital outcome of patients undergoing coronary endarterectomy: comparison between off-pump vs on pump CABG.

BACKGROUND: Due to advancement of non-surgical methods of coronary revascularization the patients referred for surgery have extensive and complex coronary anatomy. Patients with diffuse atheromatous coronary artery disease required coronary artery reconstruction or coronary endarterectomy (CE). Coronary endarterectomy on beating heart needs skill and better surgical technique. Coronary endarterectomy along with coronary artery bypass grafting (CABG) done on beating heart is compared with coronary endarterectomy done by using conventional CABG technique. METHODS: Seven hundred and ninety five consecutive patients underwent CABG from January 2006 to March 2007 in a prospective randomized trial at cardiac surgery department, Punjab Institute of Cardiology, Lahore; out of these 115 patients underwent coronary endarterectomy (CE) and were included in this study. RESULTS: Coronary artery bypass grafting was performed in 115 patients. Seventy two (62.6%) were in group A on-pump and 43 (37.39%) were in group B off-pump. Mean age in group A was 55.68 +/- 1.06 and 52.63 +/- 1.40 in group B. Sixty six male and 6 female were included in group A, 40 male and 3 female patients were in group B. In-hospital mortality among patients undergoing CABG was 5.6 % in on-pump group and 2.3 % in off-pump group (p = 0.649), the duration of post-operative mechanical ventilation in on-pump was 6.78 +/- 9.34 hours and 5 +/- 4.0 hours in off-pump group (p = 0.060), 66.7% patients in on-pump and 58.1% patients in off-pump group required blood transfusions, Intra-aortic balloon pump (IABP) was required in 5.6% of the patients in on-pump group. Other factors included, smoking 26.4% in on-pump and 41.9% in off-pump group (p = 0.01), Intensive care unit (ICU) stay was statistically significant 4 +/- 3 in on-pump group and 4 +/- 2 in off-pump group (p = 0.02), and drain in on-pump group was 455 +/- 208 ml and 540 +/- 370 ml in off-pump group (p = 0.01). CONCLUSION: Coronary endarterectomy (CE) has higher post-operative morbidity and mortality but the post-operative outcome after the procedure on either technique is comparable and CE is feasible on off-pump technique as well.

Short-term fasting attenuates the response of the HPG axis to kisspeptin challenge in the adult male rhesus monkey (Macaca mulatta).

AIMS: In primates, changes in nutritional status affect the hypothalamic-pituitary-gonadal (HPG) axis by still poorly understood mechanisms. Recently, hypothalamic kisspeptin-GPR54 signaling has emerged as a significant regulator of this neuroendocrine axis. The present study was designed to examine whether suppression of the reproductive function by acute food-restriction in a non-human primate is mediated by decreased responsiveness of the HPG axis to endogenous kisspeptin drive. MAIN METHODS: Five intact adult male rhesus monkeys habituated to chair-restraint, received intravenous boli of human kisspeptin-10 (KP10, 50 microg), hCG (50 IU), and vehicle (1 ml) in both fed and 48-h fasting conditions. Plasma concentrations of glucose, cortisol and testosterone (T) were measured by using enzymatic and specific RIAs, respectively. KEY FINDINGS: The acute 48-h fasting decreased plasma glucose (P<0.01) and T (P<0.005) levels, and increased cortisol levels (P<0.05). KP10 administration caused a robust stimulation of T secretion in both fed and fasted monkeys. However, mean T concentration and T AUC after KP10 administration were significantly (P<0.01-0.005) reduced in fasted monkeys. Likewise, the time of the first significant increase in post-KP10 T levels was also significantly (P<0.01) delayed. T response to hCG stimulation was similar in fed and fasted monkeys. SIGNIFICANCE: The present results indicate that under fasting conditions the KP10 induced T response is delayed and suppressed. These data support the notion that fasting-induced suppression of the HPG axis in the adult male rhesus monkey may involve, at least in part, a reduction in the sensitivity of the GnRH neuronal network to endogenous kisspeptin stimulation.