•Modified mineral supply may affect bone mineralisation differently between sites.•Bone demineralisation occurred within 2 weeks and recovery within 4 weeks.•Bone demineralisation and recovery ...followed similar trends for all body sites.•Bone mineralisation of lumbar spine was most sensitive to dietary mineral changes.•Head or front leg may serve as proxies for assessing whole body bone mineralisation.
Skeleton bones, distinguished by trabecular and cortical bone tissue content, exhibit varied growth and composition, in response to modified dietary calcium and phosphorus levels. The study investigated how gilts adapt their individual bone and bone region mineralisation kinetics in response to changing intake of Ca and P. A total of 24 gilts were fed according to a two-phase (Depletion (D) 60–95 and Repletion (R) 95–140 kg BW, respectively). During the D phase, gilts were fed either 60% (D60) or 100% (D100) of the estimated P requirement. Subsequently, during the R phase, half of the gilts from each D diet were fed either 100% (R100) or 160% (R160) of the estimated P requirement according to a 2 × 2 factorial arrangement. Bone mineral content (BMC) was assessed in the whole body, individual bones (femur and lumbar spine L2–L4), and bone regions (head, front legs, trunk, pelvis, femur, and hind legs) every 2 weeks using dual-energy X-ray absorptiometry (DXA). At 95 kg BW, gilts fed D60 showed reduced BMC and BMC/BW ratio in all studied sites compared to those fed D100 (P < 0.001). During the depletion phase, the allometric BW-dependent regressions slopes for BMC of D100 gilts remained close to 1 for all sites and did not differ from each other. In contrast, the slopes were lower in D60 gilts (P < 0.05), with an 18% reduction in the whole body, except for the front and hind legs, femur, and pelvis, which exhibited higher reductions (P < 0.05). At 140 kg BW, BMC and BMC/BW ratio of all studied sites were similar in gilts previously fed D60 and D100, but higher in R160 than in R100 gilts (P < 0.05), except for front and hind legs. During the repletion phase, the allometric BW dependent regressions slopes for BMC were lower (P < 0.05) in R100 than in R160 gilts (for whole body −10%; P < 0.01) except for front and hind legs, femur, and pelvis. In conclusion, bone demineralisation and recovery followed similar trends for all measured body sites. However, the lumbar spine region was most sensitive whereas the hind legs were least sensitive. These data suggest that using bone regions such as the head and forelegs that can be collected easily at the slaughterhouse may be a viable alternative to whole body DXA measurement.
Testicular sperm extraction (TESE) is often an effective method for sperm retrieval from men with non-obstructive azoospermia. However, TESE has been a blind procedure that does not identify the ...focal sperm-producing areas of the testicle until after tissue has been excised from the patient. Experience with a new technique of microdissection of testicular tubules is presented here that identifies sperm-containing regions before their removal. Identification of spermatogenically active regions of the testicle is possible by direct examination of the individual seminiferous tubules. The underlying concept for this technique is simple: seminiferous tubules containing many developing germ cells, rather than Sertoli cells alone, are likely to be larger and more opaque than tubules without sperm production. In a sequential series of TESE cases for men with non-obstructive azoospermia, the ability to find spermatozoa increased from 45% (10/22) to 63% (17/27) after introduction of the microdissection technique. Microdissected samples yielded an average of 160 000 spermatozoa per sample in only 9.4 mg of tissue, whereas only 64 000 spermatozoa were found in standard biopsy samples that averaged 720 mg in weight (P < 0.05 for all comparisons). For men where microdissection was attempted, successful identification of enlarged tubules was possible in 56% (15/27) of cases. However, spermatozoa were retrieved with microdissection TESE for six men in whom sperm retrieval was unsuccessful with standard TESE approaches (35% of all men with spermatozoa retrieved). These findings suggest that microdissection TESE can improve sperm retrieval for men with non-obstructive azoospermia over that achieved with previously described biopsy techniques.
An increasing number of studies are now reporting the effects of ocean acidification on a broad range of marine species, processes and systems. Many of these are investigating the sensitive early ...life-history stages that several major reviews have highlighted as being potentially most susceptible to ocean acidification. Nonetheless there remain few investigations of the effects of ocean acidification on the very earliest, and critical, process of fertilization, and still fewer that have investigated levels of ocean acidification relevant for the coming century. Here we report the effects of near-future levels of ocean acidification (≈−0.35 pH unit change) on sperm swimming speed, sperm motility, and fertilization kinetics in a population of the Pacific oyster Crassostrea gigas from western Sweden. We found no significant effect of ocean acidification – a result that was well-supported by power analysis. Similar findings from Japan suggest that this may be a globally robust result, and we emphasise the need for experiments on multiple populations from throughout a species' range. We also discuss the importance of sound experimental design and power analysis in meaningful interpretation of non-significant results.
Abstract
STUDY QUESTION
Do human Sertoli cells in testes that exhibit the Sertoli cell-only (SCO) phenotype produce substantially less glial cell line-derived neurotrophic factor (GDNF) than Sertoli ...cells in normal testes?
SUMMARY ANSWER
In human SCO testes, both the amounts of GDNF mRNA per testis and the concentration of GDNF protein per Sertoli cell are markedly reduced as compared to normal testes.
WHAT IS KNOWN ALREADY
In vivo, GDNF is required to sustain the numbers and function of mouse spermatogonial stem cells (SSCs) and their immediate progeny, transit-amplifying progenitor spermatogonia. GDNF is expressed in the human testis, and the ligand-binding domain of the GDNF receptor, GFRA1, has been detected on human SSCs. The numbers and/or function of these stem cells are markedly reduced in some infertile men, resulting in the SCO histological phenotype.
STUDY DESIGN, SIZE, AND DURATION
We determined the numbers of human spermatogonia per mm2 of seminiferous tubule surface that express GFRA1 and/or UCHL1, another marker of human SSCs. We measured GFRA1 mRNA expression in order to document the reduced numbers and/or function of SSCs in SCO testes. We quantified GDNF mRNA in testes of humans and mice, a species with GDNF-dependent SSCs. We also compared GDNF mRNA expression in human testes with normal spermatogenesis to that in testes exhibiting the SCO phenotype. As controls, we also measured transcripts encoding two other Sertoli cell products, kit ligand (KITL) and clusterin (CLU). Finally, we compared the amounts of GDNF per Sertoli cell in normal and SCO testes.
PARTICIPANTS/MATERIALS SETTING METHODS
Normal human testes were obtained from beating heart organ donors. Biopsies of testes from men who were infertile due to maturation arrest or the SCO phenotype were obtained as part of standard care during micro-testicular surgical sperm extraction. Cells expressing GFRA1, UCHL1 or both on whole mounts of normal human seminiferous tubules were identified by immunohistochemistry and confocal microscopy and their numbers were determined by image analysis. Human GDNF mRNA and GFRA1 mRNA were quantified by use of digital PCR and Taqman primers. Transcripts encoding mouse GDNF and human KITL, CLU and 18 S rRNA, used for normalization of data, were quantified by use of real-time PCR and Taqman primers. Finally, we used two independent methods, flow cytometric analysis of single cells and ELISA assays of homogenates of whole testis biopsies, to compare amounts of GDNF per Sertoli cell in normal and SCO testes.
MAIN RESULTS AND THE ROLE OF CHANCE
Normal human testes contain a large population of SSCs that express GFRA1, the ligand-binding domain of the GDNF receptor. In human SCO testes, GFRA1 mRNA was detected but at markedly reduced levels. Expression of GDNF mRNA and the amount of GDNF protein per Sertoli cell were also significantly reduced in SCO testes. These results were observed in multiple, independent samples, and the reduced amount of GDNF in Sertoli cells of SCO testes was demonstrated using two different analytical approaches.
LARGE SCALE DATA
N/A
LIMITATIONS, REASONS FOR CAUTION
There currently are no approved protocols for the in vivo manipulation of human testis GDNF concentrations. Thus, while our data suggest that insufficient GDNF may be the proximal cause of some cases of human male infertility, our results are correlative in nature.
WIDER IMPLICATIONS OF THE FINDINGS
We propose that insufficient GDNF expression may contribute to the infertility of some men with an SCO testicular phenotype. If their testes contain some SSCs, an approach that increases their testicular GDNF concentrations might expand stem cell numbers and possibly sperm production.
STUDY FUNDING/COMPETING INTEREST(S)
This research was funded by the Eunice Kennedy Shriver National Institute for Child Health and Human Development, National Centers for Translational Research in Reproduction and Infertility Program (NCTRI) Grant 1R01HD074542-04, as well as grants R01 HD076412-02 and P01 HD075795-02 and the U.S.-Israel Binational Science Foundation. Support for this research was also provided by NIH P50 HD076210, the Robert Dow Foundation, the Frederick & Theresa Dow Wallace Fund of the New York Community Trust and the Brady Urological Foundation. There are no competing interests.
•Void distribution in a narrow rectangular channel with various non-uniform inlet conditions for beyond bubbly flow conditions.•Modeling of void diffusion due to bubble collision ...phenomena.•Validation of the models in an adiabatic air-water two-phase flow.
Modeling and simulation study of industrial scale beyond-bubbly flows, like cap-bubbly, cap-turbulent and churn-turbulent, using the two-fluid model, has been limited due to the wide range of interfacial length scales involved and lack of physics-based models for bubble hydrodynamics and interaction mechanisms for such flow conditions. One important feature of beyond bubbly flow is the existence of a wide range of bubble sizes and shapes and thus different transport characteristics. The accuracy of two-fluid model predictions is strongly dependent on the constitutive relations used for the two-fluid model. The objectives of this proposed work are the following: select a set of physics-based constitutive relations and implement these models into CFX; evaluate these models for a wide range of test conditions in cap-turbulent and churn-turbulent flows. The interfacial structure is dynamically evaluated using bubble interaction mechanisms (coalescence and break up) from the two-group Interfacial Area Transport Equation implemented as source and sink terms into ANSYS CFX. The assessments for the validity of the models were carried out in comparison to data collected at Purdue University for two-phase flow in a narrow, rectangular duct. Non-uniform inlet conditions were applied in order to evaluate phase diffusion models in CFX. A new turbulence-induced bubble collision diffusion model for the two-group bubbles system has been implemented in ANSYS CFX. The CFD results predict the measured data to within the expected error, correctly capturing the transverse redistribution of phases due to diffusion for both uniform and non-uniform inlet phase distributions. This illustrates the importance of bubble collision phenomena in correctly predicting phase distributions in a channel.
BACKGROUND: Y chromosome microdeletions are associated with severe male factor infertility. In this study, the success rate of testicular sperm retrieval was determined for men with deletions of AZF ...regions a, b or c. METHODS: AZF deletions were detected by PCR of 30 sequence‐tagged sites within Yq emphasizing the AZFa, b and c regions. Semen analysis and diagnostic testis biopsy or testicular sperm extraction (TESE) findings were correlated with the specific AZF region deleted. RESULTS: A total of 78 men with AZF deletions included three with AZFa deletion, 11 with AZFb, 42 with AZFc, 16 with AZFb+c and six with Yq (AZFa+b+c). All men with AZFa, AZFb, AZFb+c and Yq deletions were azoospermic and no sperm were found with TESE or biopsy. Of men with isolated AZFc deletion, sperm were found in 75% (9/12) by TESE and 45% (9/20) on biopsy (56% overall); 62% (26/42) were azoospermic and 38% (16/42) severely oligozoospermic. A total of 7 patients with deletion patterns that included the complete AZFa region and 23 that included the complete AZFb region who underwent TESE or biopsy did not have sperm detected by these surgical measures. CONCLUSIONS: Microdeletion of the entire AZFa or AZFb regions of the Y chromosome portends an exceptionally poor prognosis for sperm retrieval, whereas the majority of men with AZFc deletion have sperm within the semen or testes available for use in IVF/ICSI.
Zinc (Zn) is essential for swine and poultry and native Zn concentrations in feedstuffs are too low to meet their Zn requirement. Dietary Zn bioavailability is affected by phytate, phytase and Zn ...supplemented in organic form is considered as more bioavailable than inorganic sources. A meta-analysis using GLM procedures was processed using broiler and piglet databases to investigate, within the physiological response of Zn, (1) the bioavailability of inorganic and organic Zn sources (Analysis I); (2) the bioavailability of native and inorganic Zn dependent from dietary phytates, vegetal and supplemental phytase activity (Analysis II). Analysis I: the bioavailability of organic Zn relative to inorganic Zn sources ranged, depending on the variable, from 85 to 117 never different from 100 (P > 0.05). The coefficients of determination of the regressions were 0.91 in broilers and above 0.89 in piglets. Analysis II: in broilers, bone Zn was explained by supplemental Zn (linear and quadratic, P < 0.001) and by supplemental phytase (linear, P < 0.001). In piglets, the interaction between dietary Zn and phytates/phytases was investigated by means of a new variable combining dietary phytic phosphorus (PP) and phytase activity. This new variable represents the remaining dietary PP after its hydrolysis in the digestive tract, mainly due to phytase and is called non-hydrolyzed phytic phosphorus (PP(NH)). Bone Zn was increased with native Zn (P < 0.001), but to a lower extent in high PP or low phytase diets (ZN(N) × PP(NH), P < 0.001). In contrast, the increase in bone zinc in response to supplemental Zn (P < 0.001) was not modulated by PP(NH) (P > 0.05). The coefficients of determination of the regressions were 0.92 in broilers and above 0.92 in piglets. The results from the two meta-analyses suggest that (1) broilers and piglets use supplemented Zn, independent from Zn source; (2) broiler use native Zn and the use is slightly enhanced with supplemental phytase; (3) however, piglets are limited in the use of native Zn because of the antagonism of non-hydrolyzed dietary phytate. This explains the higher efficacy of phytase in improving Zn availability in this specie.
Immune recovery was retrospectively analyzed in a cohort of 41 patients with acute leukemia, myelodysplastic syndrome and nonmalignant diseases, who received αβ T- and B-cell-depleted allografts from ...haploidentical family donors. Conditioning regimens consisted of fludarabine or clofarabine, thiotepa, melphalan and serotherapy with OKT3 or ATG-Fresenius. Graft manipulation was carried out with anti-TCRαβ and anti-CD19 Abs and immunomagnetic microbeads. The γδ T cells and natural killer cells remained in the grafts. Primary engraftment occurred in 88%, acute GvHD (aGvHD) grades II and III-IV occurred in 10% and 15%, respectively. Immune recovery data were available in 26 patients and comparable after OKT3 (n=7) or ATG-F (n=19). Median time to reach >100 CD3+ cells/μL, >200 CD19+ cells/μL and >200 CD56+ cells/μL for the whole group was 13, 127 and 12.5 days, respectively. Compared with a historical control group of patients with CD34+ selected grafts, significantly higher cell numbers were found for CD3+ at days +30 and +90 (267 vs 27 and 397 vs 163 cells/μL), for CD3+4+ at day +30 (58 vs 11 cells/μL) and for CD56+ at day +14 (622 vs 27 cells/μL). The clinical impact of this accelerated immune recovery will be evaluated in an ongoing prospective multicenter trial.
•Required time to recover from bone mineral deficit was verified in replacement gilt.•Within 2 weeks, bone mineral of 60 kg gilt decreased by deficient dietary Ca and P.•Within 4 weeks, bone mineral ...of 100 kg gilt recovered by adequate dietary Ca and P.•Within 2 weeks, bone mineral of 100 kg gilt recovered by excessive dietary Ca and P.•Finisher diet without mineral P was no concern for late selected replacement gilt.
This study investigated the ability of replacement gilts to adapt their calcium and phosphorus utilization and their kinetics in bone mineralization to compensate for modified intake of these nutrients by applying a novel Ca and P depletion and repletion strategy. A total of 24 gilts were fed according to a two-phase feeding program. In the first phase, gilts (60–95 kg BW) were fed ad libitum a depletion diet providing either 60% (D60; 1.2 g digestible P/kg) or 100% (D100; 2.1 g digestible P/kg) of the estimated P requirement. In the second phase, gilts (95–140 kg BW) were fed restrictively (aim: 700–750 g/d BW gain) a repletion diet. Half of the gilts from each depletion diet were randomly assigned to either a control diet or a high-P diet (R100 and R160; with 2.1 and 3.5 g digestible P/kg, respectively) according to a 2 × 2 factorial design, resulting in four treatments: D60-R100, D60-R160, D100-R100 and D100-R160. Dual-energy X-ray absorptiometry was used to measure whole-body bone mineral content (BMC), bone mineral density (BMD) and lean and fat tissue mass on each gilt at 2-week intervals. The depletion and repletion diets, fed for 5 and 8 weeks, respectively, did not influence growth performance. The D60 gilts had a reduced BMC and BMD from the second week onwards and ended (95 kg BW) with 9% lower values than D100 gilts (P < 0.001). During repletion, D60 gilts completely recovered the deficit in bone mineralization from the second and fourth week onwards, when fed R160 (D60-R160 vs D100-R160) or R100 (D60-R100 vs D100-R100) diets, respectively (treatment × time interaction, P < 0.001); thus, the depletion diets did not affect these values at 140 kg BW. These results illustrate the rapid homeostatic counter-regulation capacity of dietary Ca and P, and they show the high potential to limit dietary digestible P concentration by completely excluding the use of mineral phosphates during the depletion phase, representative of the fattening period, without causing any detrimental effects to gilts at mating. The gilts were able to recover their BMC deficit between their selection at 95 kg BW and first mating at 140 kg BW by increasing their dietary Ca and P efficiency. Finally, excess dietary digestible P, requiring increased amounts of mineral phosphates, further increased the gilts’ BMC.
Immune recovery was retrospectively analyzed in a cohort of 41 patients with acute leukemia, myelodysplastic syndrome and nonmalignant diseases, who received alpha beta T- and B-cell-depleted ...allografts from haploidentical family donors. Conditioning regimens consisted of fludarabine or clofarabine, thiotepa, melphalan and serotherapy with OKT3 or ATG-Fresenius. Graft manipulation was carried out with anti-TCR alpha beta and anti-CD19 Abs and immunomagnetic microbeads. The gamma delta T cells and natural killer cells remained in the grafts. Primary engraftment occurred in 88%, acute GvHD (aGvHD) grades II and III-IV occurred in 10% and 15%, respectively. Immune recovery data were available in 26 patients and comparable after OKT3 (n=7) or ATG-F (n=19). Median time to reach >100 CD3+ cells/ mu L, >200 CD19+ cells/ mu L and >200 CD56+ cells/ mu L for the whole group was 13, 127 and 12.5 days, respectively. Compared with a historical control group of patients with CD34+ selected grafts, significantly higher cell numbers were found for CD3+ at days +30 and +90 (267 vs 27 and 397 vs 163 cells/ mu L), for CD3+4+ at day +30 (58 vs 11 cells/ mu L) and for CD56+ at day +14 (622 vs 27 cells/ mu L). The clinical impact of this accelerated immune recovery will be evaluated in an ongoing prospective multicenter trial.
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