Germ cell-specific disruption of the Meig1 gene causes impaired spermiogenesis in mice.

Meiosis expressed gene 1 (Meig1) was originally identified in a search for mammalian genes potentially involved in meiosis. Seven mouse Meig1 transcripts with the same coding region, but different 5'-UTRs, have been identified. These transcripts have different tissue distributions, two are only present in the testis. In the testis, Meig1 is present in germ cells and Sertoli cells. A Meig1 conditional knockout model has been generated. When Meig1 was inactivated globally by crossing with Cmv-Cre transgenic mice, the Meig1-deficient males were sterile due to severe spermiogenic defects, and had no obvious defects in meiosis. To further study its role in individual cell types in the testis, the Meig1(flox) mice were crossed with Hsp2a-Cre, Prm-Cre, and Amh-Cre mice, in which the Cre recombinase is driven by the heat shock protein 2 (Hsp2a) gene promoter (expressed in spermatocytes), the protamine 1 gene promoter (expressed in post-meiotic spermatids) and the anti-Mullerian hormone (Amh) gene promoter (expressed in Sertoli cells) respectively. Both Meig1 mRNA and protein were undetectable in testis of the Hsp2a-Cre; Meig1(flox/flox) mice and all the mutant adult males tested were sterile. This phenotype mirrors that of the Cmv-Cre; Meig1(flox/flox) mice. Even though the total testicular Meig1 mRNA and protein expression levels were dramatically reduced in testis of the Prm-Cre; Meig1(flox/flox) males, all the mice tested were fertile, and there was no significant difference in sperm count and sperm motility compared with age-matched Meig1(flox/flox) male mice. Disruption of Meig1 in the Sertoli cells did not affect the MEIG1 protein expression. Amh-Cre; Meig1(flox/flox) males were fertile, and produced the same amount of spermatozoa as age-matched Meig1(flox/flox) mice. The testicular histology was also normal. Our results indicate that MEIG1 regulates spermiogenesis through effects in germ cells alone, and that the Meig1 gene must be active during a discrete period in spermatogenesis after which it is dispensable.

Retinal changes in various altitude illnesses.

INTRODUCTION: This study aimed to evaluate the retinal changes associated with altitude illness in young soldiers. METHODS: A total of 50 young soldiers with altitude illness, who were referred to a tertiary care hospital between October 2003 and January 2006, were included in the study. RESULTS: All the soldiers were male. The mean age of the subjects was 30.3 (range 20-44) years. Nine (18 percent) soldiers had acute mountain sickness (AMS), nine (18 percent) had high-altitude pulmonary oedema (HAPE) and 20 (40 percent) had high-altitude cerebral oedema (HACE). Retinal haemorrhages were observed in 29 soldiers (58 percent). Among these 29 cases, two also had symptoms of AMS, five had symptoms of HAPE and twelve had symptoms of HACE. High-altitude retinal haemorrhage alone as a sign of altitude illness was seen in ten cases. Severe grades of high-altitude retinopathy were found mostly in soldiers who suffered from HAPE and HACE. Visual recovery was complete in 22 (76 percent) soldiers. High-altitude retinal haemorrhage was associated with partial visual impairment in five (17 percent) soldiers and permanent visual loss in two (seven percent) soldiers. CONCLUSION: Retinal haemorrhages were noticed in 58 percent of soldiers with altitude illness. The association of severe grades of high-altitude retinopathy with HAPE and HACE was statistically significant. Branch retinal vein occlusion with macular oedema is an additional finding in our study, which has not been previously reported.

Tear Break-up Time in High Altitude Areas.

BACKGROUND: In high altitude areas, patients report with irritation, redness and foreign body sensation in their eyes suggesting tear film abnormality due to low humidity and windy environmental conditions. METHODS: Tear Break- up Time (TBUT) was studied in 100 subjects consisting of local population and those originally from plains residing in high altitude areas in Ladakh. RESULT: There were 24% individuals with TBUT of < 5 seconds, 30% between 6-10 seconds and 46% with TBUT of > 10 seconds. In symptomatic patients with TBUT of <5 seconds, eight cases had irritation of eyes, six foreign body sensation and two cases had pain, watering, irritation and redness of eyes. Pterygium was seen in 12 individuals and inter palpebral congestion in 4 cases. CONCLUSION: A total of 24% cases showed abnormal (<5 seconds) TBUT. Abnormality of tear film in the presence of low humidity and windy condition with high ultraviolet radiation may lead to ocular discomfort and pterygium in these areas.

External Versus Endoscopic Dacryocystorhinostomy: A Retrospective Study.

BACKGROUND: External dacryocystorhinostomy (DCR) has been the standard surgery for nasolacrimal duct obstruction before the development of endoscopic DCR. METHODS: This retrospective study included 103 patients with 109 cases (6 cases with bilateral disease) of nasolacrimal duct obstruction, of which 55 were treated with external DCR and 54 with endoscopic DCR. They were followed up for a minimum period of six months for surgical outcome. RESULT: The mean age of the patients in both the groups was 30 years. The success rate defined as absence of epiphora in the external DCR group was 90.9% and 95 % in the endoscopic DCR group. Majority of the recurrence, 5 out of the 6 (83.3%) in the external DCR group occurred in the first two months of surgery, while one reported four months after the surgery. The problem of intraoperative hemorrhage encountered in the external DCR group was not faced in the endoscopic DCR group. Revision of DCR was indicated in 9.1 % of cases in external DCR group as compared to 5.5 % in the endoscopic group. Serious complications like keloid formation and cosmetically unacceptable scar were not noticed in the external DCR group. Cerebrospinal fluid (CSF) rhinorrhoea was not encountered in the endoscopic group. CONCLUSION: Both the external DCR and the endoscopic DCR are effective surgical approaches for nasolacrimal duct obstruction with comparable success rate.

Conjunctival-Limbal Autograft for Primary and Recurrent Pterygium.

BACKGROUND: It has been postulated that pterygium results from hypo function of limbal stem cells. Therefore conjunctival-limbal autograft has been advocated for the treatment of this condition. This study was undertaken to evaluate the results of conjunctival-limbal autograft procedure in primary and recurrent pterygia. METHODS: 32 eyes of 28 individuals with primary and recurrent pterygium (24 primary, 8 recurrent) were undertaken for conjunctival-limbal autograft procedure under peribulbar anaesthesia followed by topical antibiotic- steroid drops for two weeks. The cases were reviewed as per protocol for 6 to 18 months. RESULT: There was no recurrence of pterygium in these cases and they were free from any major postoperative complications. CONCLUSION: Conjunctival-limbal autograft is the procedure of choice for primary and recurrent pterygia.

Validation of a testis specific serine/threonine kinase [TSSK] family and the substrate of TSSK1 & 2, TSKS, as contraceptive targets.

A family of testis specific serine/threonine kinases, TSSK1-4 and SSTK, in addition to the substrate of TSSK1 & 2, TSKS, have been studied during the past several years in our laboratory. This paper will provide a general background on these kinases through review of pertinent literature and then will summarize data from our laboratory germane to evaluating these kinases as candidate targets for future development of small molecule kinase inhibitors that may serve to regulate male fertility. Bio-informatic and structural analyses of human TSSK1-4 and SSTK indicate that these kinases constitute a unique subfamily belonging to the AMPK branch on the human kinome tree. Expression studies showed that all five kinases and the TSKS substrate are testis abundant, if not strictly testis specific, indicating that tissue specific contraceptive targeting is possible. In situ hybridization further confirmed that mouse TSSK2, SSTK and TSKS are post-meiotic in their expression patterns, a finding that makes them possible targets of reversible contraceptive intervention by preserving spermatogonia and spermatocytes. Our laboratory detected TSSK2, TSKS and SSTK proteins in mature spermatozoa for the first time. TSKS was localized to the centrioles of human spermatozoa, while TSSK2 was observed in the sperm neck, equatorial segment and mid-piece of the sperm tail, and SSTK was localized in the equatorial segment. The interaction and binding between human TSSK2 and TSKS was confirmed by several methods: this substrate and enzyme interaction offers a particularly interesting opportunity for drug design. In vitro kinase assay showed phosphorylation of TSKS by TSSK2. The TSKS phosphopeptide, HGLSPATPIQGCSGPPGS*PEEPPR, was identified by IMAC-LC-FTMS, with serine 285 being phosphorylated (representend by asterisk). These results provide a rationale for high-throughput screening of inhibitors for TSKS phosphorylation and further studies of members of this kinase family as targets for both male contraception and intra-vaginal spermicides.

Evidence for the involvement of proline-directed serine/threonine phosphorylation in sperm capacitation.

To become fertilization competent, mammalian sperm undergo changes in the female reproductive tract termed capacitation. Capacitation correlates with an increase in tyrosine phosphorylation; however, less is known about the role of serine/threonine phosphorylation in this process. Proline-directed phosphorylation is one of the major regulatory phosphorylation events in many cellular processes such as cell proliferation and differentiation. Using mitotic phosphoprotein monoclonal-2 (MPM-2) antibody in this study, we observed that several mouse sperm proteins in the range of 70-250 kDa underwent increased serine/threonine-proline phosphorylation during capacitation. In contrast to the time course of tyrosine phosphorylation, proline-directed phosphorylation could be observed at shorter time points of sperm incubation, and it was found to be independent of NaHCO(3) and adenosine 3'5'-cyclic monophosphate (cAMP). Similar to the regulation of the increase in tyrosine phosphorylation, cholesterol acceptors such as bovine serum albumin (BSA) or 2-hydroxypropyl-beta-cyclodextrin (2-OH-propyl-beta-CD) were essential for the regulation of proline-directed phosphorylation in mouse sperm. Furthermore, it was also found to be BSA dependent in human sperm. Among proline-directed kinases, extracellular signal-regulated kinase 1/2 (ERK1/2) is present in mammalian sperm; nevertheless, U0126 and PD098059, two inhibitors of the ERK pathway, did not block this phosphorylation in mouse sperm. In conclusion, capacitation is associated with an increase in proline-directed phosphorylation linked to cholesterol efflux in the sperm.

Manual Small Incision Cataract Surgery: Experience at a Military Hospital.

BACKGROUND: Small incision cataract surgery came into practice with the advent of phacoemulsification. However, manual small incision cataract surgery (SICS) is a useful alternative for those who do not have access to phacoemulsification machine. METHOD: A total of 69 cases of cataract were undertaken for manual incision cataract surgery and intra ocular lens (IOL) implantation using 6mm straight incision. The surgical technique and postoperative results are compared with the results of phacoemulsification and IOL implantation. RESULT: Average postoperative astigmatism was ± 0.75 dioptres. Postoperative uncorrected visual acuity (UCVA) of 6/18 or better was observed in 51(71.9%) cases after first week of the surgery. CONCLUSION: The study concludes that both phacoemulsification and small incision cataract surgery with intraocular lens (IOL) implantation are effective methods. However SICS with IOL implantation is a useful alternative in the absence of phacoemulsification machine.

Protein tyrosine phosphorylation, hyperactivation and progesterone-induced acrosome reaction are enhanced in IVF media: an effect that is not associated with an increase in protein kinase A activation.

Sperm capacitation is a prerequisite for successful in vitro fertilization (IVF) and therefore a focus of sperm preparation in IVF laboratories. The technology of IVF is, therefore, potentially valuable in advancing our understanding of the molecular processes that occur during sperm capacitation. We have investigated sperm capacitation induced by a commercial IVF medium compared to that occurring in standard capacitating medium (CM) typically used in a nonclinical setting. Percoll-washed spermatozoa were resuspended in Cook Sydney IVF medium, Cook Sydney IVF sperm buffer, Earle's balanced salt medium (capacitating medium) or a modified Earle's balanced salt medium [non-capacitating medium (NCM)] for up to 120 min at 37 degrees C and, if applicable, in the presence of 5% CO2 in air. Sperm protein kinase A (PKA) activity, PKA-dependent serine/threonine phosphorylation, tyrosine phosphorylation, hyperactivation and progesterone-induced acrosome reaction were evaluated. IVF medium was shown to accelerate sperm capacitation (compared with capacitating medium) as determined by tyrosine phosphorylation, sperm hyperactivation and progesterone-induced acrosome reaction. This effect was not associated with enhanced activation of PKA or increased levels of serine/threonine phosphorylation. In contrast, IVF sperm buffer (used for sperm preparation) did not stimulate sperm capacitation when incubated for up to 90 min. We have shown that different capacitating media vary strikingly in their efficacy and that this difference reflects activation of a pathway other than the well-characterized activation of soluble adenylyl cyclase/cAMP/PKA.

Role of signaling pathways in regulating the capacitation of mammalian spermatozoa.

Mammalian testicular spermatozoa are immotile and incompetent for fertilization. They acquire motility during epididymal maturation and fertilizing ability during a second phase of maturation in the female reproductive tract, termed as capacitation. Capacitation was discovered independently by Austin and Cang in early 1950s and was defined as the obligate period of residency of spermatozoa in the female reproductive tract, which confers on the spermatozoa the ability to fertilize an oocyte. Over the years, the definition of capacitation has changed and it has been recognized as a complex phenomenon, which is correlated with changes associated with the spermatozoa in the female tract. These alterations in metabolism, intracellular ion concentration, membrane fluidity, intracellular pH, cAMP concentration and concentration of reactive oxygen species, ultimately make the spermatozoa fertilization-competent. The molecular basis of capacitation is poorly understood despite the fact that it is an important event preceding fertilization. This review presents our current understanding of the signaling events involved in the process of capacitation.

Further experiences of Küntscher's nailing for tibial shaft fractures.

We present the experience of managing 429 fractures of the tibia fixed with straight Küntscher rods. The overall impression is quite encouraging because this method involves a short and easy operation with limited exposure and provides sound fixation. Complications have been negligible.