Charles H Muller | University of Washington School of Medicine (original) (raw)

Papers by Charles H Muller

Methods in molecular biology, Aug 9, 2012

In some cases, human spermatozoa to be used for in vitro fertilization are processed from testicu... more In some cases, human spermatozoa to be used for in vitro fertilization are processed from testicular or epididymal biopsies collected in the clinic or operating room. An appropriately equipped Andrology or Embryology Laboratory is required. Sterility must be maintained at all stages from collection and transport to identification and processing to insemination or cryopreservation. The technologist must be able to properly process and identify spermatozoa from aspirates, seminiferous tubules or pieces of testicular tissue. Recovery of undamaged spermatozoa from tubules or tissue requires mincing, squeezing, or vortexing the tissue, usually without the need of enzymatic digestion. A motility stimulant such as Pentoxifylline is commonly used to calculate the number of functionally competent spermatozoa. After recovery, spermatozoa may be used immediately for IVF-ICSI, incubated overnight prior to IVF-ICSI, or cryopreserved for future use. Methods for identifying, purifying, and determining the number and motility of spermatozoa during these processes are presented.

Biology of Reproduction, 1982

We have generated a monoclonal antibody directed against an antigenic determinant appearing on th... more We have generated a monoclonal antibody directed against an antigenic determinant appearing on the surface of mouse sperm tails during passage through the epididymis (a determinant that we now term sperm maturation antigen number four [SMA 41). The present study demonstrates that sperm retained in the ductuli efferentes following ligation do not acquire the antigen, suggesting that its appearance is not due to changes intrinsic to the sperm, but that the epididymal environment is required. To examine the role of the epididymis in the appearance of this antigen, sections of unfixed frozen or fixed, paraffin embedded tissue from different regions of the male reproductive tract have been studied by indirect immunofluorescence. Results indicate that the antigen is a secretory product of the epididymal epithelium, produced in a short segment of the distal caput epididymidis. Ligation experiments show that absence of sperm or testicular fluid from the epididymis does not affect production of this antigen. Examination of prepubertal mice indicates that antigen production is age dependent, production beginning in the epididymis of mice between 2 and 4 weeks of age. Indirect immunofluorescence analysis of sections of a variety of tissues and organs shows that the antigen is restricted to sperm and to epithelial cells of the male reproductive tract. Finally, experiments comparing the antibody-induced agglutinability of sperm from the caput epididymidis to that of sperm from the cauda epididymidis gives further evidence that the antigen resides on the sperm surface.

PLOS Biology, 2021

Male germ cell (GC) production is a metabolically driven and apoptosis-prone process. Here, we sh... more Male germ cell (GC) production is a metabolically driven and apoptosis-prone process. Here, we show that the glucose-sensing transcription factor (TF) MAX-Like protein X (MLX) and its binding partner MondoA are both required for male fertility in the mouse, as well as survival of human tumor cells derived from the male germ line. Loss of Mlx results in altered metabolism as well as activation of multiple stress pathways and GC apoptosis in the testes. This is concomitant with dysregulation of the expression of male-specific GC transcripts and proteins. Our genomic and functional analyses identify loci directly bound by MLX involved in these processes, including metabolic targets, obligate components of male-specific GC development, and apoptotic effectors. These in vivo and in vitro studies implicate MLX and other members of the proximal MYC network, such as MNT, in regulation of metabolism and differentiation, as well as in suppression of intrinsic and extrinsic death signaling pat...

Fertility and Sterility, 1993

Objective: To determine if one mechanism of albumin-mediated support of human sperm capacitation ... more Objective: To determine if one mechanism of albumin-mediated support of human sperm capacitation is lipid (cholesterol) transfer activity and contamination of albumin with Lipid Transfer Protein-I (LTP-I). Design and Main Outcome Measures: Measure lipid transfer activity in various bovine and human albumin preparations; relate this activity to albumin-supported capacitation (measured by zona-free hamster oocyte sperm penetration assay) and acrosome reactions; and attempt to detect LTP-I in active albumins. Remove LTP-I from albumin which supports capacitation and reassess this support. Reconstitute capacitation support by addition of purified LTP-I. Setting and Subjects: Healthy sperm donors with normal semen analyses were recruited by the Reproductive Biology-Andrology Laboratory in a university medical center. Results: Albumin preparations that effectively support capacitation have high levels of lipid transfer activity and of LTP-I, a protein responsible for lipid transfer activity. Preparations with lower levels of capacitation support have less lipid transfer activity. Removal of L TP-I from supportive albumin significantly reduces the capacitation support, and this is restored by purified LTP-I. Progesterone concentrations in these preparations are negligible. Conclusions: The variable abilities of albumin preparations to support in vitro sperm capacitation are largely dependent on the presence of contaminating LTP-I. The cholesterol transfer activity of this protein, which is present in human serum and follicular fluid, may be one mechanism in the process of capacitation.

Journal of Andrology, 1987

Sperm maturation antigen 4 (SMA-4) is a surface component of the mouse sperm tail. Previously, im... more Sperm maturation antigen 4 (SMA-4) is a surface component of the mouse sperm tail. Previously, immunofluorescence studies indicated that SMA-4 may be secreted by principal cells of the distal caput epididymidis and bound to spermatozoa as they pass through that region of the duct. In the present study, detergent extracts of spermatozoa from the cauda epididymidis were subjected to polyacrylamide gel electrophoresis under reducing and denaturing conditions, transferred to nitrocellulose, and immunostained with a monoclonal antibody against SMA-4. A band of approximately 54,000 molecular weight was revealed. The band was also stained by the periodic acid-Schiff (PAS) procedure. This glycoprotein was not detected in extracts of spermatozoa from the proximal caput epididymidis or of spermatozoa from the cauda epididymidis that were preincubated for 4 hours in an in vitro fertilization environment. Blots of sperm-free fluid from the corpus and cauda epididymidis displayed an immunoreactive and PAS-positive band of about 85,000 molecular weight that was not observed in fluid from the caput epididymidis. The difference in the molecular weights of the antigen in the fluid and that in extracts of cauda spermatozoa suggests that SMA-4 may be modified chemically upon association with the sperm surface.

JAIDS Journal of Acquired Immune Deficiency Syndromes, 2017

Introduction: HIV-1 is transmitted through semen from men to their sexual partners. Genital infec... more Introduction: HIV-1 is transmitted through semen from men to their sexual partners. Genital infections can increase HIV-1 RNA shedding in semen, but shedding also occurs in the absence of typical pathogens. We hypothesized that higher bacterial concentrations in semen would be associated with higher HIV-1 RNA levels. Methods: We analyzed semen samples from 42 HIV-1-seropositive Kenyan men using quantitative polymerase chain reaction (PCR) to assess bacterial concentrations and real-time PCR to measure HIV-1 RNA levels. Generalized estimation equations were used to evaluate associations between these 2 measures. Broad-range 16S rRNA gene PCR with pyrosequencing was performed on a subset of 13 samples to assess bacterial community composition. Results: Bacteria were detected in 96.6% of 88 samples by quantitative PCR. Semen bacterial concentration and HIV-1 RNA levels were correlated 0.30 (P = 0.01). The association between bacterial concentration and HIV-1 RNA detection was not significant after adjustment for antiretroviral therapy (ART) (adjusted odds ratio: 1.27, 95% CI: 0.84 to 1.91). Factors associated with semen bacterial concentration included insertive anal sex (adjusted beta 0.92, 95% CI: 0.12 to 1.73) and ART use (adjusted beta: 20.77, 95% CI: 21.50 to 0.04). Among 13 samples with pyrosequencing data, Corynebacterium spp., Staphylococcus spp., and Streptococcus spp. were most frequently detected. Conclusion: Most of these HIV-1-infected men had bacteria in their semen. ART use was associated with undetectable semen HIV-1 RNA and lower semen bacterial concentrations, whereas insertive anal sex was associated with higher bacterial concentrations. Additional studies evaluating the relationship between semen bacteria, inflammation, mucosal immunity, and HIV-1 shedding are needed to understand implications for HIV-1 transmission.

Cell Differentiation, 1982

Proceedings of the 1976 Laurentian Hormone Conference, 1977

Publisher Summary It has long been recognized that the control of gonadal function by pituitary h... more Publisher Summary It has long been recognized that the control of gonadal function by pituitary hormones (gonadotropins) is a general feature of vertebrate reproductive physiology. Numerous studies on the hormones of eutherian mammals have established the existence of two chemically distinct types of gonadotropin molecules in the pituitary-luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Recent biochemical studies on these molecules have revealed that each is a glycoprotein consisting of two chemically non-identical subunits, designated α and β. The physiological actions of the two gonadotropins in mammals are still subject to intensive investigation; however, it is agreed that FSH and LH have somewhat different roles in the regulation of gonadal function. This chapter describes a flow diagram of the protocol for the fractionation of pituitary hormones, with particular reference to gonadotropins and growth hormone (GH), from various nonmammalian species. The strong resemblance in chromatographic behavior among the fractions identified as FSH and LH from most nonmammalian species and their mammalian counterparts provided preliminary evidence of chemical similarity among the different species of FSH and of LH. Biochemical analyses on the six highly purified species of LH and FSH (chicken, turkey, alligator, snapping turtle, sea turtle, and bullfrog) yielded additional evidence for the homologies between mammalian and nonmammalian hormones.

A potentially important event during sperm capacitation is the loss of sperm membrane cholesterol... more A potentially important event during sperm capacitation is the loss of sperm membrane cholesterol. Although the exact mechanisms mediating this loss are not known, albumin and high density lipoprotein have been proposed as lipid acceptors. The authors propose that lipid transfer may be involved in capacitation as a specific mediator in the sequence of events leading to sperm membrane cholesterol loss. We present the first direct evidence of lipid transfer activity (LTA) in human follicular fluid (HFF). The redistribution of 14C-cholesteryl ester among human plasma lipoproteins was used as a measure of LTA (% Transfer [%T]). The HFF was fractionated by S-300 gel filtration chromatography and assayed for LTA. Three peaks of activity were consistently eluted from the column. Each peak of LTA also stimulated human sperm to penetrate zona-free hamster oocytes after short capacitating incubations. The peak with highest LTA (12.75 +/- 1.11%T) with an Mr approximately 68,000, gave the greatest stimulation (penetration index, PI: 3.34 +/- 0.96 fold increase above control, n = 4). The HFF also showed a significant dose response for both LTA and PI, whereas bovine serum albumin did not. These results demonstrate the existence of LTA in HFF and suggest that a specific lipid transfer protein may have a role in human sperm capacitation or acrosome reaction.

Archives of Andrology, 1985

The males of 279 infertile couples were evaluated with hamster ovum sperm penetration assay (SPA)... more The males of 279 infertile couples were evaluated with hamster ovum sperm penetration assay (SPA) and seminal fluid analysis. The mean SPA score for the total population was 23.0% penetration with a range of 0-97%. Twenty five percent of the patients demonstrated scores within the abnormal range (0-10%), and 15% were in the "equivocal" range (11-14%). Comparing each individual with the total population using linear regression analysis, it was noted that sperm concentration, percent motility, and percent oval forms varied directly with the SPA, and the slopes of the relationships are positive and statistically significant (p less than 0.0001, 0.002, and 0.0001, respectively). The relationship between SPA and volume is not statistically significant (p greater than or equal to 0.354). To determine whether the SPA could be utilized to establish appropriate normal parameters for various components of SFA, these were analyzed in 169 men who had SPAs of greater than or equal to 15%. Although most SFA values fell within the normal range for this group, there were several exceptions, particularly with respect to percent motility and the presence of leukocytes in the semen. Comparing the percentage of males with abnormal SPA in groups of couples with or without a demonstrable abnormality affecting fertility in the wife, no statistically significant differences could be found. The value of the SPA and SFA in investigating males of infertile couples is discussed.

Seminars in Reproductive Medicine, 2013

Advances in diagnostic techniques and therapies have drastically improved cancer survival rates. ... more Advances in diagnostic techniques and therapies have drastically improved cancer survival rates. However, the testes are highly susceptible to chemotherapy and radiation therapy, and spermatogenesis and semen parameters can be negatively impacted either permanently or transiently. Fertility preservation is an important issue to patients, and sperm banking has a positive psychological effect that helps patients cope emotionally with cancer. Innovations in sperm freezing and assisted reproductive technologies are improving the fecundity of males regardless of sperm parameters, and now even allow for fertility potential after gonadotoxic therapy. Experimental options also exist for prepubertal males, and hold promise for use in the future. At present, sperm cryopreservation is underutilized in the setting of a cancer diagnosis, and pretreatment referrals to fertility specialists are inconsistently offered. A multidisciplinary team approach is critical to educate families on the available options and help accomplish fertility preservation in a timely manner.

The Journal of Urology, 2012

sperm cell extracts by Western blot, ASC and caspase-1 were found to be elevated in SCI subjects ... more sperm cell extracts by Western blot, ASC and caspase-1 were found to be elevated in SCI subjects compared to controls. The expression of these two proteins was further confirmed by immunocytochemistry with confocal microscopy. Caspase-1 was located in the head and the midpiece whereas ASC was located mainly in the proximal section of the sperm tail. CONCLUSIONS: This study provides the first evidence of the inflammasome in human semen. The involvement of caspase-1 and ASC suggests an immunologic basis for abnormal semen quality in men with SCI. Inflammasome inactivation may be a possible target for therapeutic intervention.

International Journal of Gynecology & Obstetrics, 1990

Bacteria can be isolated from most seminal fluid samples, but the significance of these microorga... more Bacteria can be isolated from most seminal fluid samples, but the significance of these microorganisms is uncertain because most men lack symptoms associated with bacterial infection of the reproductive tract. We obtained semen samples from 37 men attending a Special Infertility Clinic and assessed the relationship between seminal fluid microorganisms and seminal fluid analysis including sperm motility, morphology, and concentration; the numbers of polymorphonuclear leukocytes and other white blood cells; and the hamster zona-free oocyte sperm penetration assay. Aerobic and/or anaerobic bacteria were recovered from 36 of the 37 samples. One hundred eighty-eight isolates (113 aerobes, 74 anaerobes, and one yeast) were recovered, with a mean of 5.2 isolates per semen specimen. The microorganisms recovered from the samples included: coagulase-negative staphylococci (89%), viridans streptococci (65%), diphtheroids (86%), Peptostreptococcus sp (62%), Bacteroides sp (27%), Gardnerella vaginalis (19%), Lactobacillus sp (16%), Actinomyces sp (16%), Enterococcus (11%), and Veillonella (11%). Other microorganisms including group B streptococcus, Hemophilus, Escherichia coli, Mobihincus, and Clostridium were each recovered from fewer than 10% of the specimens. When the microbiology of seminal fluid specimens with or without polymorphonuclear leukocytes was compared, the presence of polymorphonuclear leukocytes in the semen was not associated with the isolation of staphylococci (33 versus 25%), viridans streptococci (33 versus 28%), Bacteroides sp (17 versus 37%), or Peptostreptococcus (31 versus 33%) (P>.05 for each comparison). The proportion of semen samples yielding bacterial isolates was similar after categorization by normal motility (more than 60%), pyospermia (six or more leukocytes per 100 sperm), sperm concentration, morphology, and a normal sperm penetration assay (11% or more). Likewise, the median numbers of isolates per specimen were similar for each group. These observations suggest that bacteriospermia is not associated with either pyospermia or abnormal sperm function and probably represents bacterial colonization rather than active infection.

Human Reproduction Update, 2013

† Introduction † Methods † Factors affecting male reproductive capacity † Regulatory aspects of r... more † Introduction † Methods † Factors affecting male reproductive capacity † Regulatory aspects of reproductive toxicology † Assessing male reproductive capacity † Sperm function: physiology regulation and factors affecting it † Assessments of sperm toxicity † Testing for sperm toxicity † Conclusions and summary of recommendations background: Male reproductive potential continues to be adversely affected by many environmental, industrial and pharmaceutical toxins. Pre-emptive testing for reproductive toxicological (side-)effects remains limited, or even non-existent. Many products that come into direct contact with spermatozoa lack adequate testing for the absence of adverse effects, and numerous products that are intended for exposure to spermatozoa have only a general assumption of safety based on the absence of evidence of actual harm. Such assumptions can have unfortunate adverse impacts on at-risk individuals (e.g. couples who are trying to conceive), illustrating a clear need for appropriate up-front testing to establish actual 'sperm safety'. methods: After compiling a list of general areas within the review's scope, relevant literature and other information was obtained from the authors' personal professional libraries and archives, and supplemented as necessary using PubMed and Google searches. Review by co-authors identified and eliminated errors of omission or bias. results: This review provides an overview of the broad range of substances, materials and products that can affect male fertility, especially through sperm fertilizing ability, along with a discussion of practical methods and bioassays for their evaluation. It is concluded that products can only be claimed to be 'sperm-safe' after performing objective, properly designed experimental studies; extrapolation from supposed predicate products or other assumptions cannot be trusted. conclusions: We call for adopting the precautionary principle, especially when exposure to a product might affect not only a couple's fertility potential but also the health of resulting offspring and perhaps future generations.

General and Comparative Endocrinology, 1977

General and Comparative Endocrinology, 1980

Testes of amphibians representing three anuran and four urodelan families were examined for their... more Testes of amphibians representing three anuran and four urodelan families were examined for their abilities to secrete androgens in vitro in response to highly purified amphibian (Rana) and ovine LH or FSH. A relatively high specificity for LHs over FSHs was found for Rana catesbeiana, Ambystoma iigrinum, and Boliroglossa species. For Rana, testis pieces and dispersed testis cells responded similarly. Ovine and homologous Ambystoma LHs were highly active in Ambystoma, relative to the respective FSHs, and Rana LH was moderately more potent than Rana FSH. In contrast, androgen secretion by testes of Bufo marinus and Pseudoeurycea smithi was elicited by both gonadotropins; LH appeared to be somewhat more potent than FSH, but dose-response curves were not parallel. In Xenopus laevis, ovine gonadotropins were equally potent, and only a slight specificity for Rana LH over FSH was observed. For Taricha species, FSHs were generally equally potent to, or more potent than LHs. Homologous pituitary homogenate, but not Rana or ovine gonadotropins, stimulated androgen secretion by Necturus maculosus testes. Although LH specificity was observed in the two species in which homologous gonadotropins were tested (Rana and Ambystoma), the results generally do not support the hypothesis of LH specificity for testicular androgen secretion in Amphibia.

Biology of Reproduction, 1988

The fibrous sheath is a unique cytoskeletal component in the principal-piece segment sections of ... more The fibrous sheath is a unique cytoskeletal component in the principal-piece segment sections of mouse, rat, and hamster testis. The antigen was not detected on Western blots of mouse brain, kidney, liver, or thymus. These results indicate that A TC recognizes a protein integral to the fibrous sheath of the principal piece of sperm detected by immunohistochemistry late in spermiogenesis that is probably restricted to the male germ cell line.

Fertility and Sterility, 2022

OBJECTIVE To examine the association between cultivable vaginal Lactobacillus and fecundability i... more OBJECTIVE To examine the association between cultivable vaginal Lactobacillus and fecundability in Kenyan women attempting nonmedically assisted conception. DESIGN Prospective preconception cohort. SETTING Nairobi and Mombasa, Kenya. PATIENT(S) Women trying to conceive who reported ≤3 months of pre-enrollment conception attempt time. INTERVENTION(S) Cultivable Lactobacillus (primary), Lactobacillus morphotypes on Gram stain (secondary). MAIN OUTCOME MEASURE(S) Participants reported the first day of their last menstrual period and recent sexual behavior, underwent pregnancy testing, and provided vaginal specimen samples for Lactobacillus culture and Gram stain at ≤6 monthly preconception visits. The outcome was fecundability-the per-menstrual cycle probability of pregnancy. Associations between cultivable Lactobacillus and Lactobacillus morphotypes on Gram stain at the visit before each pregnancy test and fecundability were estimated using proportional probabilities models to generate fecundability ratios (FRs). RESULT(S) A total of 458 women contributed 1,376 menstrual cycles. At enrollment, 65.3% (n = 299) of participants had cultivable Lactobacillus, 47.4% (n = 217) had cultivable hydrogen peroxide producing Lactobacillus, and 64.6% (n = 296) had Lactobacillus detected on Gram stain. In unadjusted analysis, there was no association between cultivable Lactobacillus at the prior visit and fecundability (FR, 0.92; 95% CI, 0.73-1.16); results were similar after adjustment for age, frequency of condomless sex, and study site (adjusted FR, 0.92; 95% CI, 0.72-1.18). Lactobacillus on Gram stain at the visit prior was associated with modestly higher fecundability (adjusted FR, 1.18; 95% CI, 0.92-1.51). CONCLUSION(S) Cultivable Lactobacillus was not associated with fecundability, although Lactobacillus morphotypes detected on Gram stain were somewhat associated with increased fecundability. The relationship between vaginal Lactobacillus and fecundity may be species-specific.

F&S Reviews, 2021

Albumin, a vital protein in cell culture systems, is derived from whole blood or blood products. ... more Albumin, a vital protein in cell culture systems, is derived from whole blood or blood products. The culture of human gametes and developing embryos for assisted reproductive technology (ART) uses albumin of human origin. Human serum albumin (HSA) is derived from expired blood obtained from blood banks. This blood has been stored in polyvinyl chloride bags made clear and flexible with di-2-ethylhexyl phthalate (DEHP). However, DEHP can leach from the bags into stored blood and cofractionate with HSA during albumin isolation. DEHP and its metabolite, mono-ethylhexyl phthalate, are known endocrine disruptors that are reported to have negative effects when directly supplemented in media for in vitro fertilization using gametes from a variety of animals. Therefore, the contamination of ART media with DEHP and mono-ethylhexyl phthalate through HSA supplementation may affect the outcomes of ART procedures. Although the embryology laboratory is strictly monitored to prevent a wide variety of contaminations, phthalate contamination of HSA has not been broadly examined. This review outlines the function of HSA in ART procedures and the production of HSA from whole blood. Finally, the review highlights the effects of acute phthalate exposures on gametes during in vitro procedures. Phthalates found in human serum albumin are present in media used for ART at levels that impair developmental endpoints in model species.