The limitations of testicular organoids: are they truly as promising as we believe?

Organoid systems have revolutionised various facets of biological research by offering a three-dimensional (3D), physiologically relevant in vitro model to study complex organ systems. Over recent years, testicular organoids have been publicised as promising platforms for reproductive studies, disease modelling, drug screening, and fertility preservation. However, the full potential of these systems has yet to be realised due to inherent limitations. This paper offers a comprehensive analysis of the current challenges associated with testicular organoid models. Firstly, we address the inability of current organoid systems to fully replicate the intricate spatial organisation and cellular diversity of the in vivo testis. Secondly, we scrutinise the fidelity of germ cell maturation within the organoids, highlighting incomplete spermatogenesis and epigenetic inconsistencies. Thirdly, we consider the technical challenges faced during organoid culture, including nutrient diffusion limits, lack of vasculature, and the need for specialised growth factors. Finally, we discuss the ethical considerations surrounding the use of organoids for human reproduction research. Addressing these limitations in combination with integrating complementary approaches, will be essential if we are to advance our understanding of testicular biology and develop novel strategies for addressing reproductive health issues in males.

SPERM FACTORS AND EGG ACTIVATION: Phospholipase C zeta (PLCZ1) and the clinical diagnosis of oocyte activation deficiency.

Oocyte activation deficiency (OAD) remains the predominant cause of total/low fertilization rate in assisted reproductive technology. Phospholipase C zeta (PLCZ1) is the dominant sperm-specific factor responsible for triggering oocyte activation in mammals. OAD has been linked to numerous PLCZ1 abnormalities in patients experiencing failed in vitro fertilization or intracytoplasmic sperm injection cycles. While significant efforts have enhanced our understanding of the clinical relevance of PLCZ1, and the potential effects of genetic variants upon functionality, our ability to apply PLCZ1 in a diagnostic or therapeutic role remains limited. Artificial oocyte activation is the only option for patients experiencing OAD but lacks a reliable diagnostic approach. Immunofluorescence analysis has revealed that the levels and localization patterns of PLCZ1 within sperm can help us to indirectly diagnose a patient's ability to induce oocyte activation. Screening of the gene encoding PLCZ1 protein is also critical if we are to fully determine the extent to which genetic factors might play a role in the aberrant expression and/or localization patterns observed in infertile patients. Collectively, these findings highlight the clinical potential of PLCZ1, both as a prognostic indicator of OAD and eventually as a therapeutic agent. In this review, we focus on our understanding of the association between OAD and PLCZ1 by discussing the localization and expression of this key protein in human sperm, the potential genetic causes of OAD, and the diagnostic tools that are currently available to us to identify PLCZ1 deficiency and select patients that would benefit from targeted therapy.

The use of autologous platelet-rich plasma (PRP) versus no intervention in women with low ovarian reserve undergoing fertility treatment: a non-randomized interventional study.

PURPOSE: To investigate the impact of a 3-month course of intracortical injections of autologous platelet-rich plasma (PRP) upon ovarian reserve markers versus no intervention in women with low ovarian reserve prior to undergoing assisted reproductive technology (ART). METHODS: Prospective controlled, non-randomized comparative study conducted in a private fertility clinic, in Venezuela. Women with abnormal ovarian reserve markers (FSH, AMH and AFC) who declined oocyte donation were allocated to one of the following groups according to patient choice: monthly intracortical ovarian PRP injections for three cycles, or no intervention. Primary outcomes were the change in FSH, AMH and AFC pre- and post-treatment. Secondary outcomes included the number of oocytes collected and fertilized, biochemical/clinical pregnancy rates and miscarriage and live birth rates. RESULTS: Eighty-three women were included, of which 46 received PRP treatment and 37 underwent no intervention. Overall median age was 41 years (IQR 39-44). There were no demographic differences between the study groups. At the 3-month follow-up, women treated with PRP experienced a significant improvement in FSH, AMH and AFC, whereas there was no change in the control group. Furthermore, overall rates of biochemical (26.1% versus 5.4%, P = 0.02) and clinical pregnancy (23.9% versus 5.4%, P = 0.03) were higher in the PRP group, while there was no difference in the rates of first trimester miscarriage and live birth between groups. CONCLUSION: PRP injections are effective and safe to improve markers of low ovarian reserve prior to ART, although further evidence is required to evaluate the impact of PRP on pregnancy outcomes.

Effects of oral antioxidant treatment upon the dynamics of human sperm DNA fragmentation and subpopulations of sperm with highly degraded DNA.

The primary aim of this study was to determine the effect of oral antioxidant treatment (1500 mg of l-Carnitine; 60 mg of vitamin C; 20 mg of coenzyme Q10; 10 mg of vitamin E; 10 mg of zinc; 200 µg of vitamin B9; 50 µg of selenium; 1 µg of vitamin B12) during a time period of 3 months upon the dynamics of sperm DNA fragmentation following varying periods of sperm storage (0 h, 2 h, 6 h, 8 h and 24 h) at 37 °C in a cohort of 20 infertile patients diagnosed with asthenoteratozoospermia. A secondary objective was to use the sperm chromatin dispersion test (SCD) to study antioxidant effects upon a specific subpopulation of highly DNA degraded sperm (DDS). Semen parameters and pregnancy rate (PR) were also determined. Results showed a significant improvement of DNA integrity at all incubation points (P < 0.01). The proportion of DDS was also significantly reduced (P < 0.05). Semen analysis data showed a significant increase in concentration, motility, vitality and morphology parameters. Our results suggest that antioxidant treatment improves sperm quality not only in terms of key seminal parameters and basal DNA damage, but also helps to maintain DNA integrity. Prior administration of antioxidants could therefore promote better outcomes following assisted reproductive techniques.

Oocyte activation, phospholipase C zeta and human infertility.

BACKGROUND: Mammalian oocytes are activated by intracellular calcium (Ca(2+)) oscillations following gamete fusion. Recent evidence implicates a sperm-specific phospholipase C zeta, PLCζ, which is introduced into the oocyte following membrane fusion, as the responsible factor. This review summarizes the current understanding of human oocyte activation failure and describes recent discoveries linking certain cases of male infertility with defects in PLCζ expression and activity. How these latest findings may influence future diagnosis and treatment options are also discussed. METHODS: Systematic literature searches were performed using PubMed, ISI-Web of Knowledge and The Cochrane Library. We also scrutinized material from the United Nations and World Health Organization databases (UNWHO) and the Human Fertilization and Embryology Authority (HFEA). RESULTS AND CONCLUSIONS: Although ICSI results in average fertilization rates of 70%, complete or virtually complete fertilization failure still occurs in 1-5% of ICSI cycles. While oocyte activation failure can, in some cases, be overcome by artificial oocyte activators such as calcium ionophores, a more physiological oocyte activation agent might release Ca(2+) within the oocyte in a more efficient and controlled manner. As PLCζ is now widely considered to be the physiological agent responsible for activating mammalian oocytes, it represents both a novel diagnostic biomarker of oocyte activation capability and a possible mode of treatment for certain types of male infertility.

Reduced amounts and abnormal forms of phospholipase C zeta (PLCzeta) in spermatozoa from infertile men.

BACKGROUND: In mammals, oocyte activation at fertilization is thought to be induced by the sperm-specific phospholipase C zeta (PLCzeta). However, it still remains to be conclusively shown that PLCzeta is the endogenous agent of oocyte activation. Some types of human infertility appear to be caused by failure of the sperm to activate and this may be due to specific defects in PLCzeta. METHODS AND RESULTS: Immunofluorescence studies showed PLCzeta to be localized in the equatorial region of sperm from fertile men, but sperm deficient in oocyte activation exhibited no specific signal in this same region. Immunoblot analysis revealed reduced amounts of PLCzeta in sperm from infertile men, and in some cases, the presence of an abnormally low molecular weight form of PLCzeta. In one non-globozoospermic case, DNA analysis identified a point mutation in the PLCzeta gene that leads to a significant amino acid change in the catalytic region of the protein. Structural modelling suggested that this defect may have important effects upon the structure and function of the PLCzeta protein. cRNA corresponding to mutant PLCzeta failed to induce calcium oscillations when microinjected into mouse oocytes. Injection of infertile human sperm into mouse oocytes failed to activate the oocyte or trigger calcium oscillations. Injection of such infertile sperm followed by two calcium pulses, induced by assisted oocyte activation, activated the oocytes without inducing the typical pattern of calcium oscillations. CONCLUSIONS: Our findings illustrate the importance of PLCzeta during fertilization and suggest that mutant forms of PLCzeta may underlie certain types of human male infertility.

The pattern of localization of the putative oocyte activation factor, phospholipase Czeta, in uncapacitated, capacitated, and ionophore-treated human spermatozoa.

BACKGROUND: Recent studies suggest that in mammals, oocyte activation at fertilization is triggered by a sperm-specific phospholipase C, PLCzeta. We investigated PLCzeta localization in human spermatozoa. METHODS: A polyclonal antibody was generated against human PLCzeta and used in immunoblotting and immunofluorescence studies of ejaculated human sperm in uncapacitated and capacitated states. An ionophore was also used to induce the acrosome reaction in vitro. RESULTS: After verifying specificity of the anti-PLCzeta antibody by immunoblotting, immunofluorescence studies showed that the predominant localization of PLCzeta in uncapacitated sperm was in the equatorial region, a pattern maintained following capacitation and ionophore treatment. The analysis of pooled samples showed approximately 88% of uncapacitated sperm expressed PLCzeta in the equatorial region, whereas approximately 35% and approximately 21% of sperm expressed additional populations of PLCzeta in the acrosomal or post-acrosomal region, respectively. One population of PLCzeta was observed in the post-acrosomal region of approximately 12% of sperm. The proportion of cells with post-acrosomal PLCzeta increased following capacitation and ionophore treatment (P < 0.05). The same tendency was found in individual samples. There was a strong correlation (r = 0.716, P < 0.0001) between presence of an intact acrosome and proportion of sperm immunoreactive to PLCzeta in the acrosomal region. CONCLUSIONS: PLCzeta was variably detectable in three localities within the sperm head: the equatorial segment and acrosomal/post-acrosomal region. Variability in PLCzeta localization in sperm from fertile males may reflect differences in oocyte activation capabilities between individuals or within an ejaculate. This approach may help in investigating the possible links between PLCzeta and certain types of male infertility.

A quantitative study of testicular germ cell populations in masculinized neomale common carp (Cyprinus carpio L.).

The main objective of the present study was to investigate the effects of 17 alpha-methyltestosterone treatment upon the testicular germ cells of gynogenetic masculinized neomale common carp (Cyprinus carpio L.) in comparison with diploid carp. Gynogenetic common carp progeny (mean body weight, BW, 2.6+/-0.3g; mean total length, 10.4+/-0.5 cm) were treated for a period of 40 days with 17 alpha-methyltestosterone (MT) at a dose of 100mg kg(-1). The oral administration of MT resulted in 61.5-100% of fish exhibiting male gonads. The masculinized neomales exhibited reduced (P<0.05) body weight (BW=22.9+/-0.8) but significantly increased (P<0.05) mean testis weight (2.1+/-0.3) and mean gonadosomatic index (GSI=9.5+/-0.2%) in comparison with fish not treated with MT (BW 54.8+/-1.3; GSI=0.61%). Furthermore, treatment with MT also resulted in an increased number of fish exhibiting abnormal gonads. However, neomales did not exhibit abnormalities in the development of sperm ducts. MT treatment significantly increased germ cell volume, nuclear diameter, nuclear volume and cyst volume (P<0.01 in all cases) in MT-treated fish compared to untreated fish. The area occupied by seminiferous tubules, the number of Sertoli cells and germ cells per cyst, and the number of Leydig cells were significantly (P<0.05) greater in fish treated with MT. The carp neomales exhibited approximately 20-60% more Sertoli cells per cyst (P<0.05). Leydig cell nuclear volume and Leydig cell individual volume were significantly reduced in MT-treated groups (P<0.05) compared with untreated groups. In conclusion, our study strongly suggests that the abnormal gonadal structure evident in masculinized neomales could be explained by a combination of MT-induced genetic (homozygosity) and anabolic effects (upon germ and somatic cells).

In vivo gene transfer into the testis by electroporation and viral infection--a novel way to study testis and sperm function.

The study of gene function in testis and sperm has been greatly assisted by creation of transgenic mice by injection of a transgene into the fertilised egg. However this approach is costly and laborious and is not applicable to other species of importance for the study of sperm function, such as the hamster. We have investigated alternative ways of expressing transgenes in mouse and hamster testis and sperm by in vivo gene transfer. DNA expression constructs were introduced into the testis by injection of DNA followed by electroporation, or by injection of a lentiviral vector. Expression of fluorescent proteins was assessed by fluorescence microscopy. In vivo gene transfer by electroporation led to expression of a fluorescent reporter protein and a fluorescently tagged version of sperm protein phospholipase C zeta in hamster and mouse testis and epididymal sperm. In vivo gene transfer by lentiviral infection led to high level expression of a fluorescent reporter protein in male germ cells. In conclusion, in vivo gene transfer offers a novel way to study gene function in testis and sperm and may also have potential as a way of creating transgenic versions of important model organisms such as the hamster.

Phospholipase Czeta, the trigger of egg activation in mammals, is present in a non-mammalian species.

The activation of the egg to begin development into an embryo is triggered by a sperm-induced increase in intracellular egg Ca2+. There has been much controversy about how the sperm induces this fundamental developmental event, but recent studies suggest that, in mammals, egg activation is triggered by a testis-specific phospholipase C: PLCzeta. Since the discovery of PLCzeta, it has been unclear whether its role in triggering egg activation is common to all vertebrates, or is confined to mammals. Here, we demonstrate for the first time that PLCzeta is present in a non-mammalian vertebrate. Using genomic and cDNA databases, we have identified the cDNA encoding a PLCzeta orthologue in the domestic chicken that, like the mammalian isoforms, is a testis-specific gene. The chicken PLCzeta cDNA is 2152 bp in size and encodes an open reading frame of 639 amino acids. When injected into mouse oocytes, chicken PLCzeta cRNA triggers Ca2+ oscillations, indicating that it has functional properties similar to those of mammalian PLCzeta. Our findings suggest that PLCzeta may have a universal role in triggering egg activation in vertebrates.

Calcium-activated potassium channel SK1- and IK1-like immunoreactivity in injured human sensory neurones and its regulation by neurotrophic factors.

Calcium-activated potassium ion channels SK and IK (small and intermediate conductance, respectively) may be important in the pathophysiology of pain following nerve injury, as SK channels are known to impose a period of reduced excitability after each action potential by afterhyperpolarization. We studied the presence and changes of human SK1 (hSK1)- and hIK1-like immunoreactivity in control and injured human dorsal root ganglia (DRG) and peripheral nerves and their regulation by key neurotrophic factors in cultured rat sensory neurones. Using specific antibodies, hSK-1 and hIK-1-like immunoreactivity was detected in a majority of large and small/medium-sized cell bodies of human DRG. hSK1 immunoreactivity was decreased significantly in cell bodies of avulsed human DRG (n = 8, surgery delay 8 h to 12 months). There was a decrease in hIK1-like immunoreactivity predominantly in large cells acutely (<3 weeks after injury), but also in small/medium cells of chronic cases. Twenty-three injured peripheral nerves were studied (surgery delay 8 h to 12 months); in five of these, hIK1-like immunoreactivity was detected proximally but not distally to injury, whereas neurofilament staining confirmed the presence of nerve fibres in both regions. These five nerves, unlike the others, had all undergone Wallerian degeneration previously and the loss of hIK1-like immunoreactivity may therefore reflect reduced axonal transport of this ion channel across the injury site in regenerated fibres, as well as decreased expression in the cell body. In vitro studies of neonatal rat DRG neurones showed that nerve growth factor (NGF) significantly increased the percentage of hSK1-positive cells, whereas neurotrophin 3 (NT-3) and glial cell line-derived neurotrophic factor (GDNF) failed to show a significant effect. NT-3 stimulated hIK1 expression, while NGF and GDNF were ineffective. As expected, NGF increased expression of the voltage-gated sodium channel SNS1/PN3 in this system. Decreased retrograde transport of these neurotrophic factors in injured sensory neurones may thus reduce expression of these ion channels and increase excitability. Blockade of IK1-like and other potassium channels by aminopyridines (4-AP and 3,4-DAP) may also explain the paraesthesiae induced by these medications. Selective potassium channel openers are likely to represent novel therapies for pain following nerve injury.

Sodium channel beta1 and beta2 subunits parallel SNS/PN3 alpha-subunit changes in injured human sensory neurons.

Voltage-gated sodium channels consist of a pore-containing alpha-subunit and one or more auxiliary beta-subunits, which may modulate channel function. We previously demonstrated that sodium channel SNS/PN3 alpha-subunits were decreased in human sensory cell bodies after spinal root avulsion injury, and accumulated at injured nerve terminals in pain states. Using specific antibodies for immunohistochemistry, we have now detected sodium channel beta1 and beta2 subunits in sensory cell bodies within control human postmortem sensory ganglia (78% of small/medium (< or = 50 microm) and 68% of large (> or = 50 microm) cells); their changes in cervical sensory ganglia after avulsion injury paralleled those described for SNS/PN3 alpha-subunits. Our results suggest that alpha- and beta-subunits share common regulatory mechanisms, but present distinct targets for novel analgesics.

Plasticity of TTX-sensitive sodium channels PN1 and brain III in injured human nerves.

Sensory neurones co-express voltage-gated sodium channels that mediate TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) currents, which may contribute to chronic pain after nerve injury. We previously demonstrated that TTX-R channels were decreased acutely in human sensory cell bodies after central axotomy, but accumulated in nerve terminals after peripheral axotomy. We have now studied the TTX-S channels PN1 and Brain III, using specific antibodies for immunohistochemistry, in dorsal root ganglia (DRG) from 10 patients with traumatic central axotomy, nerves from 16 patients with peripheral axotomy, and controls. PN1 showed temporal changes similar to the TTX-R channels in sensory cell bodies of injured DRG. In contrast, Brain III was found only in injured nerves (not control nerves, or control/central axotomy DRG). PNI and Brain III are distinct targets for novel analgesics.

Immunolocalisation of sodium channel NaG in the intact and injured human peripheral nervous system.

The voltage-gated 'glial' sodium channel NaG belongs to a distinct molecular class within the multi-gene family of mammalian sodium channels. Originally found in central and peripheral glia, NaG has since been detected in neurons in rat dorsal root ganglia (DRG) and may play a role in Schwann cell-axon interactions. We have studied the presence of NaG-like immunoreactivity in the intact and injured human peripheral nervous system using a specific affinity-purified antibody. Nerve fibres in normal and injured peripheral nerves and normal skin exhibited intense NaG-immunoreactivity. Numerous NaG-immunoreactive nerve fibres surrounded neuronal cell bodies within postmortem control DRG, and in DRG avulsed from the spinal cord (i.e. after traumatic central axotomy). There were no significant differences in the pattern of NaG immunostaining between control and avulsed DRG, or with delay after injury. Generally, the neuronal cell bodies were only very weakly immunoreactive to NaG, indicating that the NaG immunoreactivity was predominantly in Schwann cells/myelin. In accord, we demonstrated NaG immunostaining in cultured human and rat Schwann cells, and in distal nerve after wallerian degeneration. NaG thus appears to be a useful new marker for Schwann cells in the human PNS, and a role in neuropathy deserves investigation.

Immunolocalization of SNS/PN3 and NaN/SNS2 sodium channels in human pain states.

The tetrodotoxin-resistant (TTX-R) voltage-gated sodium channel SNS/PN3 and the newly discovered NaN/SNS2 are expressed in sensory neurones, particularly in nociceptors. Using specific antibodies, we have studied, for the first time in humans, the presence of SNS/PN3 and NaN/SNS2 in peripheral nerves, including tissues from patients with chronic neurogenic pain. In brachial plexus injury patients, there was an acute decrease of SNS/PN3- and NaN/SNS2-like immunoreactivity in sensory cell bodies of cervical dorsal root ganglia (DRG) whose central axons had been avulsed from spinal cord, with gradual return of the immunoreactivity to control levels over months. In contrast, there was increased intensity of immunoreactivity to both channels in some peripheral nerve fibers just proximal to the site of injury in brachial plexus trunks, and in neuromas. These findings suggest that the expression of these sodium channels in neuronal cell bodies is reduced after spinal cord root avulsion injury in man, but that pre-synthesized channel proteins may undergo translocation with accumulation at sites of nerve injury, as in animal models of peripheral axotomy. The latter may contribute to positive symptoms, as our patients all showed a positive Tinel's sign. Nerve terminals in distal limb neuromas and skin from patients with chronic local hyperalgesia and allodynia all showed marked increases of SNS/PN3-immunoreactive fibers, but little or no NaN/SNS2-immunoreactivity, suggesting that the former may be related to the persistent hypersensitive state. Axonal immunoreactivity to both channels was similar to control nerves in sural nerve biopsies in a selection of neuropathies, irrespective of nerve inflammation, demyelination or spontaneous pain, including a patient with congenital insensitivity to pain. Our studies suggest that the best target for SNS/PN3 blocking agents is likely to be chronic local hypersensitivity.

Expression and localization of messenger ribonucleic acid for the vitellogenin receptor in ovarian follicles throughout oogenesis in the rainbow trout, Oncorhynchus mykiss.

The expression and localization of vitellogenin (VTG) receptor (VTGR) mRNA were identified throughout ovarian development in the rainbow trout, Oncorhynchus mykiss. Northern blot confirmed the presence of a transcript (approximately 3.9 kilobases [kb]) that was specific to the ovary. The expression of VTGR mRNA varied throughout ovarian development and was highest in previtellogenic ovaries and in ovaries at the onset of vitellogenesis containing ovarian follicles (OF) from 35 to 600 microm in diameter. In situ hybridization using 35S riboprobes showed that the transcription of the VTGR gene was initiated in OF measuring 45-50 microm in diameter, with transcripts being exclusively localized in the ooplasm. A dramatic increase in mRNA synthesis occurred during previtellogenic growth (OF from 50 to 200 microm); this was followed by a gradual decrease during the vitellogenic growth phase. VTGR mRNA was not detected in OF greater than 1000 microm in diameter (oocytes actively sequestering VTG). Immunocytolocalization of yolk proteins derived from VTG demonstrated that oocytes started to sequester VTG when they were around 300 microm in diameter, shortly after the time of maximal density of VTGR mRNA in the ooplasm. The timing of transcription of the VTGR gene, predominantly during previtellogenesis, suggests that the VTGR is recycled to the oocyte surface during the vitellogenic growth phase.

Molecular characterization and expression of two ovarian lipoprotein receptors in the rainbow trout, Oncorhynchus mykiss.

Partial cDNAs encoding two lipoprotein receptors were isolated and sequenced from the ovary in the rainbow trout (rt), Oncorhynchus mykiss. One of the cDNAs (rt-LPR) contained the 5 domains characteristic of receptors belonging to the low-density lipoprotein receptor superfamily. The second cDNA (rt-LP[OS]R) was similar to rt-LPR but lacked 105 base pairs encoding the O-linked sugar domain. The deduced amino acid sequences of the rt-LPR and rt-LP[OS]R had between 75% and 80% identity with very-low-density lipoprotein and vitellogenin receptors of other species. The rt-lipoprotein receptor mRNAs were approximately 3.5 kilobases in size. The rt-LPR was expressed in both the ovary and somatic tissues, whereas the rt-LP[OS]R was ovary-specific. Messenger RNA for the lipoprotein receptor(s) was expressed at high levels in both pre-vitellogenic (< 0.3 mm) and early vitellogenic (up to 1 mm) follicles. Using reverse transcription-polymerase chain reaction, expression of rt-LPR and rt-LP[OS]R mRNA was also detected in larger vitellogenic follicles (up to 2.5 mm in diameter) but not in follicles in late vitellogenesis or in ovulated eggs. The sequence, ovary specificity, and pattern of ovarian expression of the rt-LPR mRNA suggest that it encodes the receptor that mediates vitellogenin uptake into the ovary.

Comparison of fixation strengths of locking head and conventional screws, in fracture and reconstruction models.

PURPOSE: Claimed clinical advantages of the locking-head mandibular reconstruction plating system include the ability to achieve stability with fewer numbers of screws per bony segment as compared with conventional screws. The purpose of this study was to test the hypothesis that increased resistance to displacement will be obtained when using locking-head as compared with the same number of conventional screws per segment in both fracture and reconstruction models. MATERIALS AND METHODS: Eight groups were tested based on the screw number (two or four), screw type (locking-head or conventional), and fracture (bony apposition) or reconstruction model (1-cm defect). Two-dimensional beam mechanics using adult bovine ribs and the Instron machine were used to develop a load-displacement curve up to 150 N for each specimen. An osteotomy was then created and the segments were reduced, with preload (fracture model) or with a 1-cm defect (reconstruction model), and plated using the Synthes locking-head plate with either two or four bicortical locking-head (4.0-mm) or conventional (2.7-mm) screws per segment. The fixed ribs were loaded to 150 N, and the displacement was recorded. RESULTS: Locking-head screws provided superior resistance when using two screws per segment in the reconstruction model as compared with conventional screws. Minimal difference was seen between other screw types within a model. The fracture model offered significantly greater (3.1 to 3.7X) resistance to displacement than did the reconstruction model. CONCLUSIONS: Locking-head screws provided significantly increased resistance to displacement when only two screws per segment were used in the reconstruction model. When four screws per segment were used, there was no significant difference between locking-head and conventional screw types in either model. The effect of bony buttressing is significant and may explain why miniplates often fail in the atrophic mandible but are successful in the fully dentate patient.

Molecular characterisation of ovarian cathepsin D in the rainbow trout, Oncorhynchus mykiss.

In fish, cathepsin D, an aspartyl protease, is believed to mediate the processing of yolk proteins in the oocyte. Cathepsin D, therefore, is vital for the production of a viable egg. This study set out to isolate and sequence the cDNA encoding cathepsin D, and to determine the developmental expression of the message in the ovary and subsequently during embryogenesis in the rainbow trout, Oncorhynchus mykiss. The full-length trout cathepsin D cDNA is 1847 base pairs (bp) long, encoding a protein of 400 amino acids (aa). The sequence consists of a putative signal peptide of 18 aa, a prosequence extending 46 aa and a mature peptide of 336 aa. The deduced sequence of rainbow trout ovarian cathepsin D shows significant homology with cathepsin D in mammals (human; 81% aa similarity), in the chicken (80% aa similarity) and in Xenopus (74% aa similarity). Our data support the contention that the primary structure of cathepsin D is highly conserved across the vertebrate phyla, from mammals to fish. Unlike cathepsin Ds in other species, however, rainbow trout cathepsin D appears to have only one putative N-glycosylation site, rather than two. The mRNA for 'ovarian' cathepsin D was expressed in both ovarian and non-ovarian tissues (liver, muscle, spleen and testis). During the development of the ovary, the highest expression levels of cathepsin D mRNA were seen at around the onset of vitellogenesis, a time when the oocytes are starting to sequester large quantities of yolk proteins. Northern hybridisation did not detect cathepsin D mRNA in either unfertilised eggs, or in fertilised eggs until after gastrulation, indicating that there is little, if any, de novo synthesis of this message at these stages of development. However, the mRNA for cathepsin D was detectable at the eyed embryo stage, and the expression of the gene increased towards the end of embryonic development.