Two alternative methods for the retrieval of somatic cell populations from the mouse ovary.

Many modern techniques employed to uncover the molecular fundamentals underlying biological processes require dissociated cells as their starting point/substrate. Investigations into ovarian endocrinology or folliculogenesis, therefore, necessitate robust protocols for dissociating the ovary into its constituent cell populations. While in the mouse, methods to obtain individual, mature follicles are well-established, the separation and isolation of single cells of all types from early mouse follicles, including somatic cells, has been more challenging. Herein we present two methods for the isolation of somatic cells in the ovary. These methods are suitable for a range of applications relating to the study of folliculogenesis and mouse ovarian development. First, an enzymatic dissociation utilising collagenase and a temporary, primary cell culture step using neonatal mouse ovaries which yields large quantities of granulosa cells from primordial, activating, and primary follicles. Second, a rapid papain dissociation resulting in a high viability single cell suspension of ovarian somatic cells in less than an hour, which can be applied from embryonic to adult ovarian samples. Collectively these protocols can be applied to a broad array of investigations with unique advantages and benefits pertaining to both.

Signal transducer and activator of transcription (STAT) 1 and STAT3 are expressed in the human ovary and have Janus kinase 1-independent functions in the COV434 human granulosa cell line.

Ovarian granulosa cells are fundamental for oocyte maintenance and maturation. Recent studies have demonstrated the importance of members of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signalling pathway in the granulosa cell population of mouse and horse ovaries, with perturbation of JAK1 signalling in the mouse shown to impair oocyte maintenance and accelerate primordial follicle activation. The presence and role of the JAK/STAT pathway in human granulosa cells has yet to be elucidated. In this study, expression of JAK1, STAT1 and STAT3 was detected in oocytes and granulosa cells of human ovarian sections from fetal (40 weeks gestation) and premenopausal ovaries (34-41 years of age; n=3). To determine the effects of JAK1 signalling in granulosa cells, the human granulosa-like cell line COV434 was used, with JAK1 inhibition using ruxolitinib. Chemical inhibition of JAK1 in COV434 cells with 100nM ruxolitinib for 72h resulted in significant increases in STAT3 mRNA (P=0.034) and p-Y701-STAT1 protein (P=0.0117), demonstrating a role for JAK1 in modulating STAT in granulosa cells. This study implicates a conserved role for JAK/STAT signalling in human ovary development, warranting further investigation of this pathway in human granulosa cell function.

Grandmaternal smoke exposure reduces female fertility in a murine model, with great-grandmaternal smoke exposure unlikely to have an effect.

STUDY QUESTION: What effect does multigenerational (F2) and transgenerational (F3) cigarette smoke exposure have on female fertility in mice? SUMMARY ANSWER: Cigarette smoking has a multigenerational effect on female fertility. WHAT IS KNOWN ALREADY: It has been well established that cigarette smoking decreases female fertility. Furthermore, a growing body of evidence suggests that smoking during pregnancy decreases the fertility of daughters and increases cancer and asthma incidence in grandchildren and great-grandchildren. STUDY DESIGN, SIZE, DURATION: Six-week-old C57BL/6 female mice were exposed nasally to cigarette smoke or room air (controls) for 5 weeks prior to being housed with males. Females continued to be exposed to smoke throughout pregnancy and lactation until pups were weaned. A subset of F1 female pups born to these smoke and non-smoke exposed females were bred to create the F2 grandmaternal exposed generation (multigenerational). Finally, a subset of F2 females were bred to create the F3 great-grandmaternal exposed generation (transgenerational). The reproductive health of F2 and F3 females was examined at 8 weeks and 9 months. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian and oocyte quality was examined in smoke exposed and control animals. A small-scale fertility trial was performed before ovarian changes were examined using ovarian histology and immunofluorescence and/or immunoblotting analysis of markers of apoptosis (TUNEL) and proliferation (proliferating cell nuclear antigen (PCNA) and anti-Mullerian hormone (AMH)). Oocyte quality was examined using immunocytochemistry to analyze the metaphase II spindle and ploidy status. Parthenogenetic activation of oocytes was used to investigate meiosis II timing and preimplantation embryo development. Finally, diestrus hormone serum levels (FSH and LH) were quantified. MAIN RESULTS AND THE ROLE OF CHANCE: F2 smoke exposed females had no detectable change in ovarian follicle quality at 8 weeks, although by 9 months ovarian somatic cell proliferation was reduced (P = 0.0197) compared with non-smoke exposed control. Further investigation revealed changes between control and smoke exposed F2 oocyte quality, including altered meiosis II timing at 8 weeks (P = 0.0337) and decreased spindle pole to pole length at 9 months (P = 0.0109). However, no change in preimplantation embryo development was observed following parthenogenetic activation. The most noticeable effect of cigarette smoke exposure was related to the subfertility of F2 females; F2 smoke exposed females displayed significantly increased time to conception (P = 0.0042) and significantly increased lag time between pregnancies (P = 0.0274) compared with non-smoke exposed F2 females. Conversely, F3 smoke exposed females displayed negligible oocyte and follicle changes up to 9 months of age, and normal preimplantation embryo development. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: This study focused solely on a mouse model of cigarette smoke exposure to simulate human exposure. WIDER IMPLICATIONS OF THE FINDINGS: Our results demonstrate that grandmaternal cigarette smoke exposure reduces female fertility in mice, highlighting the clinical need to promote cessation of cigarette smoking in pregnant women. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Australian Research Council, National Health and Medical Research Council, Hunter Medical Research Institute, Newcastle Permanent Building Society Charitable Trust, and the University of Newcastle Priory Research Centers in Chemical Biology, Healthy Lungs and Grow Up Well. The authors declare no conflict of interest.

Non-coding RNA in Spermatogenesis and Epididymal Maturation.

Testicular germ and somatic cells express many classes of small ncRNAs, including Dicer-independent PIWI-interacting RNAs, Dicer-dependent miRNAs, and endogenous small interfering RNA. Several studies have identified ncRNAs that are highly, exclusively, or preferentially expressed in the testis and epididymis in specific germ and somatic cell types. Temporal and spatial expression of proteins is a key requirement of successful spermatogenesis and large-scale gene transcription occurs in two key stages, just prior to transcriptional quiescence in meiosis and then during spermiogenesis just prior to nuclear silencing in elongating spermatids. More than 60 % of these transcripts are then stockpiled for subsequent translation. In this capacity ncRNAs may act to interpret and transduce cellular signals to either maintain the undifferentiated stem cell population and/or drive cell differentiation during spermatogenesis and epididymal maturation. The assignation of specific roles to the majority of ncRNA species implicated as having a role in spermatogenesis and epididymal function will underpin fundamental understanding of normal and disease states in humans such as infertility and the development of germ cell tumours.

Damaging legacy: maternal cigarette smoking has long-term consequences for male offspring fertility.

STUDY QUESTION: What are the effects on fertility of cigarette smoke-induced toxicity on male offspring exposed during the gestational/weaning period? SUMMARY ANSWER: Maternal cigarette smoke exposure during the gestational/weaning period causes long-term defects in male offspring fertility. WHAT IS KNOWN ALREADY: Cigarette smoke is a well-known reproductive toxicant which is particularly harmful to both fetal and neonatal germ cells. However, recent studies suggest a significant portion of young mothers in the developed world still smoke during pregnancy. In the context of male reproductive health, our understanding of the effects of in utero exposure on offspring fertility is limited. STUDY DESIGN, SIZE, DURATION: In this study, 27 C57BL/6 5-week-old female mice were exposed via the nose-only to cigarette smoke (treatment) or 27 were exposed to room air (control) for 6 weeks before being housed with stud males to produce litters. In the treatment group, smoke exposure continued throughout mating, pregnancy and lactation until weaning of pups at 21 days post birth. Male offspring were examined at post-natal days 3, 6, 12, 21 and 98 (adult). PARTICIPANTS/MATERIALS, SETTING, METHODS: Approximately 108 maternal smoke-exposed C57BL/6 offspring and controls were examined. Spermatogenesis was examined using testicular histology and apoptosis/DNA damage was assessed using caspase immunohistochemistry and TUNEL. Sertoli cell morphology and fluctuations in the spermatogonial stem cell population were also examined using immunohistochemistry. Microarray and QPCR analysis were performed on adult testes to examine specific long-term transcriptomic alteration as a consequence of maternal smoke exposure. Sperm counts and motility, zona/oolemma binding assays, COMET analysis and mitochondrial genomic sequencing were also performed on spermatozoa obtained from adult treated and control mice. Fertility trials using exposed adult male offspring were also performed. MAIN RESULTS AND THE ROLE OF CHANCE: Maternal cigarette smoke exposure caused increased gonocyte and meiotic spermatocyte apoptosis (P < 0.01) as well as germ cell depletion in the seminiferous tubules of neonatal and juvenile offspring. Aberrant testicular development characterized by abnormal Sertoli and germ cell organization, a depleted spermatogonial stem cell population (P < 0.01), atrophic seminiferous tubules and increased germ cell DNA damage (P < 0.01) persisted in adult offspring 11 weeks after exposure. Microarray analysis of adult offspring testes associated these defects with meiotic germ cell development, sex hormone metabolism, oxidative stress and Sertoli cell signalling. Next generation sequencing also revealed a high mitochondrial DNA mutational load in the testes of adult offspring (P < 0.01). Adult maternal smoke-exposed offspring also had reduced sperm counts with spermatozoa exhibiting morphological abnormalities (P < 0.01), affecting motility and fertilization potential. Odf2, a spermatozoa flagellum component required for coordinated ciliary beating, was also significantly down-regulated (P < 0.01) in maternal smoke-exposed adult offspring, with aberrant localization along the spermatozoa flagellum. Adult maternal smoke-exposed offspring took significantly longer to impregnate control females and had a slight but significant (P < 0.01) reduction in litter size. LIMITATIONS, REASONS FOR CAUTION: This study examined only one species (mouse) using a smoking model which only simulates human cigarette smoke exposure. WIDER IMPLICATIONS OF THE FINDINGS: This study represents the first comprehensive animal model of maternal smoking on male offspring reproductive function, suggesting that exposure during the gestational/weaning period causes long-term defects in male offspring fertility. This is due to a compromised spermatogonial stem cell population resulting from gonocyte apoptosis and impaired spermatogenic development. This results in significant germ cell damage and Sertoli cell dysfunction, impacting germ cell number, tubule organization, DNA damage and spermatozoa in adult offspring. This study strengthens the current literature suggesting that maternal exposure impairs male offspring fertility, which is currently debated due to conflicting studies. STUDY FUNDING/COMPETING INTERESTS: This study was funded by the Australian Research Council, Hunter Medical Research Institute, National Health and Medical Research Council of Australia and the Newcastle Permanent Building Society Charitable Trust. The authors declare no conflict of interest.

Intracellular signalling during female gametogenesis.

Female reproductive potential is dictated by the size of the primordial follicle pool and the correct regulation of oocyte maturation and activation--events essential for production of viable offspring. Although a substantial body of work underpins our understanding of these processes, the molecular mechanisms of follicular and oocyte development are not fully understood. This review summarizes recent findings which have improved our conception of how folliculogenesis and oocyte competence are regulated, and discusses their implications for assisted reproductive techniques. We highlight evidence provided by genetically modified mouse models and in vitro studies which have refined our understanding of Pi3k/Akt and mTOR signalling in the oocyte and have discovered a role for Jak/Stat/Socs signalling in granulosa cells during primordial follicle activation. We also appraise a novel role for the metal ion zinc in the regulation of meiosis I and meiosis II progression through early meiosis inhibitor (Emi2) and Mos-Mapk signalling, and examine studies which expand our understanding of intracellular calcium signalling and extrinsic Plcζ in stimulating oocyte activation.

Scrambled and fried: cigarette smoke exposure causes antral follicle destruction and oocyte dysfunction through oxidative stress.

Cigarette smoke is a reproductive hazard associated with pre-mature reproductive senescence and reduced clinical pregnancy rates in female smokers. Despite an increased awareness of the adverse effects of cigarette smoke exposure on systemic health, many women remain unaware of the adverse effects of cigarette smoke on female fertility. This issue is compounded by our limited understanding of the molecular mechanisms behind cigarette smoke induced infertility. In this study we used a direct nasal exposure mouse model of cigarette smoke-induced chronic obstructive pulmonary disease to characterise mechanisms of cigarette-smoke induced ovotoxicity. Cigarette smoke exposure caused increased levels of primordial follicle depletion, antral follicle oocyte apoptosis and oxidative stress in exposed ovaries, resulting in fewer follicles available for ovulation. Evidence of oxidative stress also persisted in ovulated oocytes which escaped destruction, with increased levels of mitochondrial ROS and lipid peroxidation resulting in reduced fertilisation potential. Microarray analysis of ovarian tissue correlated these insults with a complex mechanism of ovotoxicity involving genes associated with detoxification, inflammation, follicular activation, immune cell mediated apoptosis and membrane organisation. In particular, the phase I detoxifying enzyme cyp2e1 was found to be significantly up-regulated in developing oocytes; an enzyme known to cause molecular bioactivation resulting in oxidative stress. Our results provide a preliminary model of cigarette smoke induced sub-fertility through cyp2e1 bioactivation and oxidative stress, resulting in developing follicle depletion and oocyte dysfunction.

Suppressor of cytokine signaling 4 (SOCS4): moderator of ovarian primordial follicle activation.

Mammalian ovarian primordial follicle activation and regulation is considered as one of the most important stages of folliculogenesis and as such requires exquisite control. Selection of quiescent follicles to enter the growing pool determines the rate of supply of maturing follicles over the female reproductive lifespan. To coordinate this process a range of positive and negative input signals contribute to determine follicle fate. This study demonstrates that the cytokine Leukemia Inhibitory Factor (LIF) activates the Janus Kinase 1/Signal Transducers and Activators of Transcription 3 (JAK1/STAT3) signaling pathway in pre-granulosa cells and positively regulates primordial follicle activation. Negative regulation of the JAK/STAT pathway is controlled by the suppressor of cytokine signaling 4 (SOCS4) protein, which target members of negative feedback loops, Cardiotrophin like Cytokine (CLC), Poly (rC) Binding Protein 1 (PCBP1), and Cytosolic Malate Dehydrogenase (MDH1) to suppress follicle growth and development.

Jumping the gun: smoking constituent BaP causes premature primordial follicle activation and impairs oocyte fusibility through oxidative stress.

Benzo(a)pyrene (BaP) is an ovotoxic constituent of cigarette smoke associated with pre-mature ovarian failure and decreased rates of conception in IVF patients. Although the overall effect of BaP on female fertility has been documented, the exact molecular mechanisms behind its ovotoxicity remain elusive. In this study we examined the effects of BaP exposure on the ovarian transcriptome, and observed the effects of in vivo exposure on oocyte dysfunction. Microarray analysis of BaP cultured neonatal ovaries revealed a complex mechanism of ovotoxicity involving a small cohort of genes associated with follicular growth, cell cycle progression, and cell death. Histomorphological and immunohistochemical analysis supported these results, with BaP exposure causing increased primordial follicle activation and developing follicle atresia in vitro and in vivo. Functional analysis of oocytes obtained from adult Swiss mice treated neonatally revealed significantly increased levels of mitochondrial ROS/lipid peroxidation, and severely reduced sperm-egg binding and fusion in both low (1.5mg/kg/daily) and high (3mg/kg/daily) dose treatments. Our results reveal a complex mechanism of BaP induced ovotoxicity involving developing follicle atresia and accelerated primordial follicle activation, and suggest short term neonatal BaP exposure causes mitochondrial leakage resulting in reduced oolemma fluidity and impaired fertilisation in adulthood. This study highlights BaP as a key compound which may be partially responsible for the documented effects of cigarette smoke on follicular development and sub-fertility.

Investigation of the expression and functional significance of the novel mouse sperm protein, a disintegrin and metalloprotease with thrombospondin type 1 motifs number 10 (ADAMTS10).

Fertilization represents the culmination of a series of complex interactions between male and female gametes. Despite advances in our understanding, the precise molecular mechanisms underlying these fundamental interactions remain largely uncharacterized. There is however growing recognition that this process requires the concerted action of multiple sperm receptors that possess affinity for complementary zona pellucida ligands and those that reside on the surface of the oolemma. Among the candidate sperm proteins that have been implicated in fertilization, those belonging to the ADAM (a disintegrin and metalloprotease) family of proteases have received considerable attention. The focus of the studies described herein has been the characterization of a closely related member of this protease family, ADAMTS10 (a disintegrin and metalloprotease with thrombospondin type 1 motifs number 10). We have demonstrated that ADAMTS10 is expressed during the later stages of mouse spermatogenesis and incorporated into the acrosomal domain of developing spermatids. During sperm maturation, the protein appears to be processed before being expressed on the surface of the peri-acrosomal region of the head. Our collective data suggest that, from this position, ADAMTS10 participates in sperm adhesion to the zona pellucida. Indeed, pre-incubation of capacitated spermatozoa with either galardin, a broad spectrum inhibitor of metalloprotease activity, or anti-ADAMTS10 antisera elicited a significant reduction in their ability to engage in zona adhesion. Overall, these studies support the notion that sperm-oocyte interactions involve considerable functional redundancy and identify ADAMTS10 as a novel candidate in the mediation of these fundamentally important events.

Comparison of laparoscopic adjustable gastric banding (LAGB) with other bariatric procedures; a systematic review of the randomised controlled trials.

BACKGROUND: Bariatric surgery can provide efficient weight loss and improvement in obesity-related co-morbidities in adults. Laparoscopic adjustable gastric banding (LAGB) comprised 30.3% of all bariatric procedures between 2009 and 2010 in the UK. This review evaluates the level 1 evidence for change in co-morbidities, quality of life (QoL) and weight provided by LAGB compared with other bariatric procedures. METHOD: Systematic literature search of MEDLINE, EMBASE and CENTRAL (1988 to May 2011) was performed. Only randomised controlled trials (RCTs) were included. Studies with non-surgical comparators, open gastric banding procedures or adolescent participants were excluded. Primary outcome was change in co-morbidities. Secondary outcomes included QoL, weight loss, complications, operation time and length of stay. RESULTS: Five RCTs met the inclusion criteria. Vertical banded gastroplasty, sleeve gastrectomy and gastric bypass were compared to LAGB. Co-morbidities were reported in two studies and QoL in one. LAGB was comparable to other procedures for both of these outcomes. All five trials showed LABG to be effective in weight loss, however all comparative procedures resulted in greater weight loss. Operative time and length of hospital stay were significantly shorter with LAGB. Short-term complications were found to be consistently lower in the LAGB group. Evidence was divided with respect to long-term complications. CONCLUSION: Co-morbidities and QoL are poorly reported and showed no difference between LAGB and other bariatric procedures. Evidence suggests that LAGB is not the most effective surgical procedure to reduce weight. LAGB is associated with lower early complications and shorter operative time and length of stay, and therefore may be preferable to patients.

Differential expression profiles of conserved Snail transcription factors in the mouse testis.

Snail transcription factors are key regulators of cellular transitions during embryonic development and tumorigenesis. The closely related SNAI1 and SNAI2 proteins induce epithelial-mesenchymal transitions (EMTs), acting predominantly as transcriptional repressors, while the functions of SNAI3 are unknown. An initial examination of Snai2-deficient mice provided evidence of deficient spermatogenesis. To address the hypothesis that Snail proteins are important for male fertility, this study provides the first comprehensive cellular expression profiles of all three mammalian Snail genes in the post-natal mouse testis. To evaluate Snail transcript expression profiles, droplet digital (dd) PCR and in situ hybridization were employed. Snai1, 2 and 3 transcripts are readily detected at 7, 14, 28 days post-partum (dpp) and 7 weeks (adult). Unique cellular expression was demonstrated for each by in situ hybridization and immunohistochemistry using Western blot-validated antibodies. SNAI1 and SNAI2 are in the nucleus of the most mature germ cell types at post-natal ages 10, 15 and 26. SNAI3 is only detected from 15 dpp onwards and is localized in the Sertoli cell cytoplasm. In the adult testis, Snai1 and Snai2 transcripts are detected in spermatogonia and spermatocytes, while Snai3 is in both germ and Sertoli cells. SNAI1 protein is evident in nuclei of spermatogonia, spermatocytes, round spermatids and elongated spermatids (Stages IX-XII). SNAI2 is present in the nuclei of spermatogonia and spermatocytes, with a faint signal detected in round spermatids. SNAI3 was detected only in Sertoli cell cytoplasm, as in juvenile testes. Additionally, colocalization of SNAI1 and SNAI2 with previously identified key binding partners, LSD1 and PRC2 complex components, provides strong evidence that these important functional interactions are conserved during spermatogenesis to control gene activity. These distinct expression profiles suggest that each Snail family member has unique functions during spermatogenesis.

Optimisation of handling, activation and assessment procedures for Bufo marinus spermatozoa.

In the present study, we investigated handling, activation and assessment procedures for cane toad (Bufo marinus) spermatozoa. Optimisation of these techniques will facilitate the maintenance of sperm viability during cryopreservation and during in vitro fertilisation (IVF) techniques in reproduction technologies for endangered species. Spermatozoa were taken from testicular macerates and assessed using plasma membrane integrity assays (live/dead stains) and quantitative scores of motility parameters. In the assessment of sperm viability using live/dead stains, there were small but significant differences in the percentage of sperm from cryopreserved samples staining positive with propidium iodide, Hoechst H33258 and Trypan blue; these differences were not large and all stains performed acceptably. Spermatozoa were activated by dilution of testicular macerates in water at one of two dilution ratios (1 : 6 or 1 : 20) with or without 0.1-5.0 mM theophylline. Sperm plasma membrane integrity (unstained spermatozoa) was unaffected by either dilution ratio (osmolarity) or theophylline concentration. However, sperm motility was significantly affected by osmolarity and theophylline concentration. The stimulation of sperm motility increased with higher theophylline concentrations and these strongly interacted with lower osmolarities through a higher dilution ratio of sperm macerates with water. Spermatozoa were exposed to increasing centrifugation forces to determine tolerance to physical stresses encountered during washing procedures. Forces between 50 and 800 g were associated with a significant reduction in motility (mean 56 +/- 3% decreasing to 27 +/- 3%), but did not affect staining. In conclusion, centrifugation should be minimised in anuran sperm washing procedures; osmotic shock associated with higher dilution ratios reduces the capacity of anuran sperm to achieve high percentages of motile sperm, leading to a likely trade-off between dilution required for activation and sperm motility to optimise IVF fertilisation rates; and optimal conditions for sperm motility after activation occur at lower dilutions of suspensions with 5.0 mM theophylline. The present study has improved protocols for the handling of anuran sperm during pre- and post-cryopreservation procedures.

Effects of the plant steroidal hormone, 24-epibrassinolide, on the mitotic index and growth of onion (Allium cepa) root tips.

The purpose of the present study was to determine the effects of the steroidal plant hormone, 24-epibrassinolide (BL), on the mitotic index and growth of onion (Allium cepa) root tips. The classical Allium test was used to gather and quantify data on the rate of root growth, the stages of mitosis, and the number of mitoses in control and BL-treated groups of onions. Low doses of BL (0.005 ppm) nearly doubled the mean root length and the number of mitoses over that of controls. Intermediate doses of BL (0.05 ppm) also produced mean root lengths and number of mitoses that were significantly greater than those of the controls. The highest dose of BL (0.5 ppm) produced mean root lengths and number of mitoses that were less than control values, but the differences were not statistically significant. Examination of longitudinally sectioned root tips produced relatively similar results. This study confirms the suppositions of previous authors who have claimed that exogenously applied BL can increase the number of mitoses in plants, but failed to show cytogenetic data. This is the first report detailing the effects of BL on chromosomes and the cell cycle.

Higher gonadotrophin surge-attenuating factor bioactivity is found in small follicles from superovulated women.

Ovine and rat pituitary bioassays for gonadotrophin surge-attenuating factor (GnSAF) were utilized to determine whether the level of GnSAF bioactivity in pooled human follicular fluid (hFF) from superovulated women varied according to follicle diameter (< or = 11 mm, 12-15 mm and 16-21 mm follicles examined using the ovine bioassay, or < or = 10 mm, 11-13 mm, 14-17 mm, 18-20 mm, 21-24 mm and > or = 25 mm follicles examined using the rat bioassay). When tested using dispersed ovine pituitary cells, GnSAF bioactivity, expressed in terms of the reduction in gonadotrophin-releasing hormone (GnRH)-induced LH secretion, was inversely related to follicle diameter (P < 0.01). In response to 5 microliters hFF/well from follicles of < or = 11, 12-15 and 16-21 mm diameter, GnRH-induced LH secretion was reduced to 40.5 +/- 6.9%, 65.2 +/- 6.6% and 83.7 +/- 7.9% of control respectively. A similar inverse relationship was observed when a second batch of hFF samples from different sized follicles was tested using rat pituitary cell monolayers. Expressing GnSAF bioactivity in terms of the dose required to suppress GnRH-induced LH secretion by rat pituitary cells to 50% of the maximal suppression observed (ED50), the three smallest follicle size pools contained the most GnSAF (ED50 values of 0.13, 2.79 and 5.36 microliters hFF/well from follicles of < or = 10, 11-13 and 14-17 mm respectively). The ED50 values for follicles of 18-20, 21-24 and > or = 25 mm were 8.81, 27.1 and 60.0 microliters hFF/well respectively. Thus hFF from follicles < or = 11 mm was over 450 times more potent than hFF from follicles > or = 25 mm in suppressing GnRH-induced LH release. The ED50 values for inhibin bioactivity (measured as the suppression of basal FSH secretion from rat pituitary monolayers) were much less variable than those for GnSAF bioactivity (between 0.85 and 0.13 microliters hFF/well). Inhibin immunoreactivity, measured by a two-site immunoradiometric assay, followed the same pattern as inhibin bioactivity with lowest concentrations in the smallest follicles (41.96 ng/ml) and highest concentrations in the three largest follicle size groups (56.48-64.48 ng/ml). The specific effects of inhibin on GnRH-induced LH and basal FSH release in these pituitary bioassays were determined by incubating culture dishes with pure recombinant human inhibin at doses of 0.025-25 ng/well. In both the sheep and rat pituitary monolayers, basal FSH was suppressed (ED50 = 0.02 and 0.16 ng/well respectively).(ABSTRACT TRUNCATED AT 400 WORDS)

British Andrology Society guidelines for the assessment of post vasectomy semen samples (2002).

The British Andrology Society guidelines for the assessment of post vasectomy semen samples recommend that initial assessment is undertaken 16 weeks post vasectomy and after the patient has produced at least 24 ejaculates. The laboratory should examine a freshly produced seminal fluid specimen by direct microscopy and if no sperm are seen the centrifugate should be examined for the presence of motile and non-motile spermatozoa. It is recommended that the clinician should give clearance after the production of two consecutive sperm free ejaculates. In cases of persistent identification of non-motile spermatozoa the referring clinician should advise the patient regarding the cessation of other contraceptive precautions. Surgeons are responsible both preoperatively and postoperatively for the counselling of couples regarding complications and the possibility of late recanalisation after clearance.

Intracellular calcium measurements in individual human sperm demonstrate that the majority can respond to progesterone.

OBJECTIVE: To determine if human spermatozoa could be immobilized and intracellular calcium measurements made on individual cells to measure what proportion can respond to P. DESIGN: Spermatozoa were loaded with Fura 2 (Sigma Chemical Co., Poole, Dorest, United Kingdom) and suspended in 10% gelatin at 37 degrees C. A thin layer of the suspension was cooled to room temperature (20 degrees C to 25 degrees C) and [Ca2+]i was measured with a fluorescence microscope equipped with dual wavelength excitation and an image analysis system. SETTING: University-based laboratory. PARTICIPANTS: Semen was obtained from four fertile donors to a donor insemination program. INTERVENTIONS: None. MAIN OUTCOME MEASURES: [Ca2+]i was calculated from the ratio of Fura 2 fluorescence excited at 340 nm and that excited at 366 nm. RESULTS: One hundred six of 114 sperm examined (93%) demonstrated a significant response to P but the size and duration of the response was variable. CONCLUSION: These data demonstrate that most sperm can respond to P.

Patterns of high tension electrical injury in children and adolescents and their management.

High tension electrical burn injury occurred in twenty-nine patients over a period of seven years, causing two deaths, and demonstrating two reasonably distinct patterns. Major surface thermal burns from involvement in an electric arc, augmented by flame burns of ignited clothing, occurred in eleven patients. By contrast, eighteen children demonstrated wounds of entrance and exit of current and varying effects of deep thermal injury along the path of the maximally conductive blood vessels and nerves of extremities. An aggressive surgical approach was taken in patients with both types of injury, with the objective of prompt identification and excision of devitalized tissues and closure of thw wound. In the deep condictuve type of injury, frozen section and subsequent histologic study served as a guide to adequacy of excision and preservation of viable tissue. Even so, amputation of fifteen extremities and four other major excisions were required in twelve patients.

The jelly envelopes and fertilization of eggs of the newt, Notophthalmus viridescens.

Fertilization in Notophthalmus viridescens is internal and involves passage of the sperm through five layers of egg jelly (J5-J1, from outermost to innermost), each of which is secreted by a discrete region of the oviduct. Polyspermy is normal. Passage of the sperm through the jelly and into the egg was studied by a technique of artificial insemination similar to natural insemination, in that undiluted fluid from the vas deferens was applied directly to eggs with various layers of jelly present, followed by flooding with water three to five minutes later. In general, successful fertilization increased as the number of jelly layers increased; jellyless coelomic eggs were not fertilizable. Sperm passage through the jelly and into the egg usually occurs within one to three minutes. Upon hydration of the jelly, barriers to sperm penetration develop in layers J5 and J3. Changes in the egg jelly thus seem to be involved in the restriction of polyspermy to a low level.

Single agent vs. combination chemotherapy for ovarian cancer.

Forty evaluable patients with advanced epithelial cancers of the ovary received chemotherapy. Twenty-seven previously untreated patients underwent a 1:1 randomization between a combination of Cytoxan, Hexamethylmelamine, and Fluorouracil (CHF) versus a single agent, L-phenylalanine mustard (L-pam). Thirteen patients previously treated with other therapies received CHF as a second-line therapy. Eighty-five percent of the patients receiving triple therapy were responders, versus 57% in the single agent group. Fifty percent of the CHF group had a complete response versus 17% in the L-pam group (p = 0.09). All patients with complete resection of less than 2 cm residual disease at primary surgery were responders, regardless of the type of therapy. Response in these patients is defined in terms of disease-free interval. The importance of maximal surgical resection in management of these cancers is discussed. Five of eight patients treated with CHF undergoing second-look operations had no evidence of disease. One of three L-pam treated patients had no evidence of disease at second-look surgery. Six of 13 patients (46%) had partial response to CHF as a second-line drug.