Mammalian Muscle Research Articles

Mediators of human ureteral smooth muscle contraction-a role for erythropoietin, tamsulosin and Gli effectors.

BackgroundUreteral contractility is a poorly understood process. Contractions have been demonstrated to occur in the smooth muscle layers of the ureter. Previous work suggests the involvement of Gli family proteins and erythropoietin (EPO) in regulating mammalian ureteral smooth muscle contraction. We sought to devise a method by which the effects of these proteins and tamsulosin on distal human ureteral tissue contractility could be investigated to better understand mechanisms regulating human ureteral function.MethodsIRB approval was obtained to procure portions of extraneous distal ureteral tissue from living donor renal transplants. Contractility was measured by placing the tissue in Krebs buffer and stimulating via a uniform electric current. Contractile force was recorded with each stimulation with and without the presence of a Gli inhibitor (GANT61) or EPO. Each ureteral specimen was subsequently fixed and tested by immunohistochemistry to determine Gli, EPO and alpha-adrenergic receptor activity.ResultsElectrical field stimulation successfully elicited contractions in the ureteral tissue. Administering tamsulosin decreased force and duration of ureteral contractions. Inhibiting Gli signaling decreased contractility and EPO decreased ureteral contractile forces within 5 minutes of administration versus untreated controls. Staining confirmed Gli1 protein and α-adrenergic receptor expression in ureteral smooth muscle and epithelial tissue with EPO receptor expression confined to the epithelial layer.ConclusionsDistal ureteral contractile forces are decreased by inhibition of Gli family proteins and the α-adrenergic receptor. EPO acts within five minutes, suggesting ion channel involvement instead of changes in gene expression. Continuing work will elucidate the role of these proteins in coordinating ureteral contractions. This has implications for the use of pharmacologic methods to address ureteral contractility and dysfunctional peristalsis during stone passage, ureteroscopy, in transplant patients and potentially to reduce symptoms from ureteral stents.

Morphological changes and functional circRNAs screening of rabbit skeletal muscle development

BackgroundThe temporal expression pattern of circular RNAs (circRNAs) across developmental stages is essential for skeletal muscle growth and functional analysis. However, there are few analyses on the potential functions of circRNAs in rabbit skeletal muscle development.ResultsInitially, the paraffin sections showed extremely significant differences in the diameter, number, area and density of skeletal muscle fibers of the fetus, child, adult rabbit hind legs (P < 0.01). Then, RNA-seq libraries of these three stages were constructed. A total of 481 differentially expressed circRNAs (DE-circRNAs) and 5,658 differentially expressed genes (DEGs) were identified. Subsequently, DE-circRNAs, whose host genes were DEGs or non-DEGs, were analyzed by GO respectively. In the fetus vs. child group, up-regulated DE-circRNAs (whose host genes were DEGs) were related to muscle fiber structure, and down-regulated ones were related to mitosis. The up-regulated DE-circRNAs (whose host genes were non-DEGs) were involved in enzyme activity, methylation and glycosylation, and the down-regulated ones were involved in mitosis and catabolism. In the fetus vs. adult group, the up-regulated DE-circRNAs (whose host genes were DEGs) were related to skeletal muscle basic structure, and the down-regulated ones were also associated with cell proliferation. But the up-regulated DE-circRNAs (whose host genes were non-DEGs) were connected with regulation of histone ubiquitination, chromatin and organelles. The down-regulated DE-circRNAs were connected with the catabolism processes. In addition, novel_circ_0022663 and novel_circ_0005489, which might have coding potential, and novel_circ_0004210 and novel_circ_0001669, which might have miRNA sponge capability, were screened out.ConclusionsIn this study, hind leg muscles of fetus, child and adult rabbits were collected for paraffin section and RNA-seq to observe the structural changes of skeletal muscle and obtain circRNA expression profiles at different stages. These data provided a catalog of circRNAs related to muscle development in New Zealand rabbits, allowing us to better understand the functional transitions in mammalian muscle development.

PAX7 Is a Sensitive Marker of Skeletal Muscle Differentiation in Rhabdomyosarcoma and Tumors With Rhabdomyosarcomatous Differentiation in the Female Genital Tract.

In the female genital tract, rhabdomyosarcoma may occur in "pure" form or as a heterologous constituent of a biphasic neoplasm such as carcinosarcoma or adenosarcoma. Discriminating rhabdomyosarcoma from its histologic mimics relies on confirmation of skeletal muscle differentiation by morphology or immunohistochemistry (IHC), which can be challenging to interpret in some cases owing to limited expression. PAX7, a transcription factor expressed in mammalian muscle progenitor cells, has been reported in up to 86% of soft tissue rhabdomyosarcomas by IHC. To determine whether PAX7 IHC could augment current approaches to identify rhabdomyosarcoma in gynecologic malignancies, we assessed PAX7, myogenin, and MyoD1 IHC on whole tissue sections from 100 gynecologic tumors: 50 with rhabdomyosarcomatous differentiation and 50 with features mimicking rhabdomyosarcoma. PAX7 expression was present in 96% (48/50) of gynecologic tumors with rhabdomyosarcomatous differentiation and was absent in all rhabdomyosarcoma mimics; it was more diffusely expressed than myogenin in 16 cases and was positive in a greater percentage of tumor cells in 28 cases. PAX7 and myogenin were typically coexpressed, and no rhabdomyosarcoma exhibited complete absence of both markers; however, 2 myogenin-negative tumors were PAX7-postive. Morphologically, PAX7 localized to the nuclei of primitive-appearing cells, whereas myogenin was observed in maturing rhabdomyoblasts including strap cells. Our findings highlight the utility of PAX7 as a complementary diagnostic marker of rhabdomyosarcomatous differentiation in gynecologic tumors. PAX7 should be used in combination with other markers of skeletal muscle differentiation, namely myogenin, and may be particularly helpful in cases where myogenin and/or MyoD1 expression is limited.

Mandibular muscle troponin of the Florida carpenter ant Camponotus floridanus: extending our insights into invertebrate Ca2+ regulation.

Ants use their mandibles for a variety of functions and behaviors. We investigated mandibular muscle structure and function from major workers of the Florida carpenter ant Camponotusfloridanus: force-pCa relation and velocity of unloaded shortening of single, permeabilized fibres, primary sequences of troponin subunits (TnC, TnI and TnT) from a mandibular muscle cDNA library, and muscle fibre ultrastructure. From the mechanical measurements, we found Ca2+-sensitivity of isometric force was markedly shifted rightward compared with vertebrate striated muscle. From the troponin sequence results, we identified features that could explain the rightward shift of Ca2+-activation: the N-helix of TnC is effectively absent and three of the four EF-hands of TnC (sites I, II and III) do not adhere to canonical sequence rules for divalent cation binding; two alternatively spliced isoforms of TnI were identified with the alternatively spliced exon occurring in the region of the IT-arm α-helical coiled-coil, and the N-terminal extension of TnI may be involved in modulation of regulation, as in mammalian cardiac muscle; and TnT has a Glu-rich C-terminus. In addition, a structural homology model was built of C.floridanus troponin on the thin filament. From analysis of electron micrographs, we found thick filaments are almost as long as the 6.8μm sarcomeres, have diameter of ~ 16nm, and typical center-to-centerspacing of ~ 46nm. These results have implications for the mechanisms by which mandibular muscle fibres perform such a variety of functions, and how the structure of the troponin complex aids in these tasks.

A microwave powered polymeric artificial muscle

Twisted and coiled polymer fibres are vastly used to fabricate artificial muscles with stroke and stress outputs rivalling those of mammalian muscles. The thermal stimulation of these fibres, however, requires a tethered power source, limiting their application as truly autonomous system. Here, a novel type of coiled polymer actuator is demonstrated where thermal stimulation is realized via microwave radiation. This helical writhe shaped actuator is made of carbon nanotube (CNT) filled highly-drawn nylon 6 fibres and offers muscle-like linear movements when microwave-irradiated. The embedded CNTs absorb the external electromagnetic energy and act as volumetric heating sources in the fibres. When stimulated via microwave radiation, the unique anisotropic thermal expansion phenomenon of highly-drawn fibres provides >8% reversible length contraction of the writhe muscle with a starting actuation speed of ~0.8%/sec. The maximum specific work output is calculated to be ~0.2 kJ/kg, which is >5 fold higher than that for natural muscle. The developed technology offers an opportunity to stimulate the state-of-art thermoresponsive artificial muscles wirelessly and/or to use them for autonomous soft robotic application.

Chronic electrical stimulation reduces reliance on anaerobic metabolism in locust jumping muscle

Chronic electrical stimulation (CES) is a well-documented method for changing mammalian muscle from more fast-twitch to slow-twitch metabolic and contractile profiles. Although both mammalian and insect muscles have many similar anatomical and physiological properties, it is unknown if CES produces similar muscle plasticity changes in insects. To test this idea, we separated Schistocerca americana grasshoppers into two groups (n = 37 to 47): one that was subjected to CES for 180 min each day for five consecutive days and one group that was not. Each group was then electrically stimulated for a single time period (0, 5, 30, 60, or 180 min) before measuring jumping muscle lactate, a characteristic of fast-twitch type fibers. At each time point, CES led to a significantly reduced jumping muscle lactate concentration. Based on similar short-term CES mammalian studies, the reduction in lactate production was most likely due to a reduced reliance on anaerobic metabolism. Thus, longer stimulation periods should result in greater aerobic enzymatic activities, altered myosin ATPase, and shift fiber types. This is the first study to use electrical stimulation to explore insect muscle plasticity and our results show that grasshopper jumping muscle responds similarly to mammalian muscle.

Can a chloride channel blocker mitigate muscle fatigue?

Can a chloride channel blocker mitigate muscle fatigue?

Gerta Vrbová, a guide and a friend for a generation of neuro-myologists - Her scientific legacies and relations with colleagues.

Gerta Sidonová - Vrbová, (Trnava, Slovakia, November 28, 1926 - London, UK, October 2, 2020) has been a key neuroscientist, who for almost half a century has contributed important findings and hypotheses on the relationships between motoneurons and skeletal muscle fibers, in particular on the differentiation and extent of plasticity of the peculiar characteristics of the different types of fibers present in mammalian muscles. This issue, Ejtm 31(1), 2021, opens with the personal obituary authored by Dirk Pette, who remember his lifelong collaboration with Gerta, describing the many molecular and metabolic events that occur by changing the pattern of activation of adult muscle fibers through neuromuscular low frequency electrical stimulation. To honor the many scientific legacies of Gerta Vrbová and her impact on a generation of researchers studying myology and managements of neuromuscular disorders I add here additional examples of Gerta’s scientific heritage and of her relations with colleagues.

Gerta Vrbová, a guide and a friend for a generation of neuro-myologists – Her scientific legacies and relations with colleagues

Gerta Sidonová - Vrbová, (Trnava, Slovakia, November 28, 1926 - London, UK, October 2, 2020) has been a key neuroscientist, who for almost half a century has contributed important findings and hypotheses on the relationships between motoneurons and skeletal muscle fibers, in particular on the differentiation and extent of plasticity of the peculiar characteristics of the different types of fibers present in mammalian muscles. This issue, Ejtm 31 (1), 2021, opens with the personal obituary authored by Dirk Pette, who remember his lifelong collaboration with Gerta, describing the many molecular and metabolic events that occur by changing the pattern of activation of adult muscle fibers through neuromuscular low frequency electrical stimulation. To honor the many scientific legacies of Gerta Vrbová and her impact on a generation of researchers studying myology and managements of neuromuscular disorders I add here additional examples of Gerta’s scientific heritage and of her relations with colleagues.

Detection of biological signals from a live mammalian muscle using an early stage diamond quantum sensor

The ability to perform noninvasive and non-contact measurements of electric signals produced by action potentials is essential in biomedicine. A key method to do this is to remotely sense signals by the magnetic field they induce. Existing methods for magnetic field sensing of mammalian tissue, used in techniques such as magnetoencephalography of the brain, require cryogenically cooled superconducting detectors. These have many disadvantages in terms of high cost, flexibility and limited portability as well as poor spatial and temporal resolution. In this work we demonstrate an alternative technique for detecting magnetic fields generated by the current from action potentials in living tissue using nitrogen vacancy centres in diamond. With 50 pT/sqrt{text {Hz}} sensitivity, we show the first measurements of magnetic sensing from mammalian tissue with a diamond sensor using mouse muscle optogenetically activated with blue light. We show these proof of principle measurements can be performed in an ordinary, unshielded lab environment and that the signal can be easily recovered by digital signal processing techniques. Although as yet uncompetitive with probe electrophysiology in terms of sensitivity, we demonstrate the feasibility of sensing action potentials via magnetic field in mammals using a diamond quantum sensor, as a step towards microscopic imaging of electrical activity in a biological sample using nitrogen vacancy centres in diamond.

Regulation of &lt;i&gt;Drosophila&lt;/i&gt; Visceral Muscle Contractions by Multiple, Indirect Octopaminergic and Glutamatergic Signaling Pathways

Mammalian visceral muscles are indirectly regulated by receptors expressed in peripheral neurons and other non-contractile cells. The complex interactions between these pathways remain poorly understood. It also remains unknown whether invertebrates use pathways similar to mammals to indirectly regulate visceral contractility. To elucidate the principles of organization for the signaling pathways responsible for visceral motility and the more general logic of aminergic neuromodulation, we are using the Drosophila oviposition circuit. We find that the receptors for the adrenergic neurotransmitter octopamine as well as the metabotropic glutamate receptor are not expressed in muscle cells, but rather, in epithelial cells that line the oviduct lumen in a regionally specific pattern and in multiple subtypes of peripheral neurons, some of which co-express mechanosensory markers. We have used live imaging to demonstrate that most of the functional effects of octopamine and glutamate are mediated by indirect pathways and that contractions of the lateral and common oviducts are regulated independently. Neurons intrinsic to the lateral oviduct indirectly activate muscle cells, and mutational and optogenetic analyses indicate that Octβ2 is required for these effects. An additional indirect pathway for lateral oviduct contractions is controlled by glutamate receptors expressed on a distinct subset of neuronal processes outside of the lateral oviduct. In addition to contractions of the lateral oviduct, octopamine regulates dilation, but this occurs in both the lateral and common oviducts and is blocked by mutation of OAMB but not Octβ2. Additional, unsuspected interactions between octopamine and glutamate include contractions of the common oviduct that requires an interaction between octopamine and with glutamate, blockade of lateral oviduct contractions by preincubation with glutamate and opposing effects of octopamine and glutamate in epithelial cells. These data indicate that multiple indirect pathways regulate the oviposition circuit similar to the mechanisms described for mammalian visceral muscles. They also uncover a complex network of neuromodulatory effects that provide insight into the regulation of behavioral circuits in other systems.

μ-Crystallin in Mouse Skeletal Muscle Promotes a Shift from Glycolytic toward Oxidative Metabolism.

μ-Crystallin, encoded by the CRYM gene, binds the thyroid hormones, T3 and T4. Because T3 and T4 are potent regulators of metabolism and gene expression, and CRYM levels in human skeletal muscle can vary widely, we investigated the effects of overexpression of Crym. We generated transgenic mice, Crym tg, that expressed Crym protein specifically in skeletal muscle at levels 2.6–147.5 fold higher than in controls. Muscular functions, Ca2+ transients, contractile force, fatigue, running on treadmills or wheels, were not significantly altered, although T3 levels in tibialis anterior (TA) muscle were elevated ~190-fold and serum T4 was decreased 1.2-fold. Serum T3 and thyroid stimulating hormone (TSH) levels were unaffected. Crym transgenic mice studied in metabolic chambers showed a significant decrease in the respiratory exchange ratio (RER) corresponding to a 13.7% increase in fat utilization as an energy source compared to controls. Female but not male Crym tg mice gained weight more rapidly than controls when fed high fat or high simple carbohydrate diets. Although labeling for myosin heavy chains showed no fiber type differences in TA or soleus muscles, application of machine learning algorithms revealed small but significant morphological differences between Crym tg and control soleus fibers. RNA-seq and gene ontology enrichment analysis showed a significant shift towards genes associated with slower muscle function and its metabolic correlate, β-oxidation. Protein expression showed a similar shift, though with little overlap. Our study shows that μ-crystallin plays an important role in determining substrate utilization in mammalian muscle and that high levels of μ-crystallin are associated with a shift toward greater fat metabolism.

Cholesterol activates BK channels by increasing KCNMB1 protein levels in the plasmalemma

Calcium-/voltage-gated, large-conductance potassium channels (BKs) control critical physiological processes, including smooth muscle contraction. Numerous observations concur that elevated membrane cholesterol (CLR) inhibits the activity of homomeric BKs consisting of channel-forming alpha subunits. In mammalian smooth muscle, however, native BKs include accessory KCNMB1 (β1) subunits, which enable BK activation at physiological intracellular calcium. Here, we studied the effect of CLR enrichment on BK currents from rat cerebral artery myocytes. Using inside-out patches from middle cerebral artery (MCA) myocytes at [Ca2+]free=30 μM, we detected BK activation in response to in vivo and in vitro CLR enrichment of myocytes. While a significant increase in myocyte CLR was achieved within 5 min of CLR in vitro loading, this brief CLR enrichment of membrane patches decreased BK currents, indicating that BK activation by CLR requires a protracted cellular process. Indeed, blocking intracellular protein trafficking with brefeldin A (BFA) not only prevented BK activation but led to channel inhibition upon CLR enrichment. Surface protein biotinylation followed by Western blotting showed that BFA blocked the increase in plasmalemmal KCNMB1 levels achieved via CLR enrichment. Moreover, CLR enrichment of arteries with naturally high KCNMB1 levels, such as basilar and coronary arteries, failed to activate BK currents. Finally, CLR enrichment failed to activate BK channels in MCA myocytes from KCNMB1-/- mouse while activation was detected in their wild-type (C57BL/6) counterparts. In conclusion, the switch in CLR regulation of BK from inhibition to activation is determined by a trafficking-dependent increase in membrane levels of KCNMB1 subunits.

Whole-mount Staining of Mouse Diaphragm Neuromuscular Junctions.

The neuromuscular junction (NMJ) is a specialized synapse that connects the terminal end of a motor neuron and a skeletal muscle fiber. Defects in NMJ cause abnormalities of neuromuscular transmission, leading to NMJ disorders. The mammalian diaphragm muscle is essential for respiration and has been widely used to study NMJ formation. Here, we provide a simple and straightforward protocol for preparing diaphragms from embryonic, neonatal, and adult mice and for subsequent NMJ staining.

Myosin 7b is a regulatory long noncoding RNA (lncMYH7b) in the human heart

Myosin heavy chain 7b (MYH7b) is an ancient member of the myosin heavy chain motor protein family that is expressed in striated muscles. In mammalian cardiac muscle, MYH7b RNA is expressed along with two other myosin heavy chains, β-myosin heavy chain (β-MyHC) and α-myosin heavy chain (α-MyHC). However, unlike β-MyHC and α-MyHC, which are maintained in a careful balance at the protein level, the MYH7b locus does not produce a full-length protein in the heart due to a posttranscriptional exon-skipping mechanism that occurs in a tissue-specific manner. Whether this locus has a role in the heart beyond producing its intronic microRNA, miR-499, was unclear. Using cardiomyocytes derived from human induced pluripotent stem cells as a model system, we found that the noncoding exon-skipped RNA (lncMYH7b) affects the transcriptional landscape of human cardiomyocytes, independent of miR-499. Specifically, lncMYH7b regulates the ratio of β-MyHC to α-MyHC, which is crucial for cardiac contractility. We also found that lncMYH7b regulates beat rate and sarcomere formation in cardiomyocytes. This regulation is likely achieved through control of a member of the TEA domain transcription factor family (TEAD3, which is known to regulate β-MyHC). Therefore, we conclude that this ancient gene has been repurposed by alternative splicing to produce a regulatory long-noncoding RNA in the human heart that affects cardiac myosin composition.

Diverse and complex muscle spindle afferent firing properties emerge from multiscale muscle mechanics.

Despite decades of research, we lack a mechanistic framework capable of predicting how movement-related signals are transformed into the diversity of muscle spindle afferent firing patterns observed experimentally, particularly in naturalistic behaviors. Here, a biophysical model demonstrates that well-known firing characteristics of mammalian muscle spindle Ia afferents - including movement history dependence, and nonlinear scaling with muscle stretch velocity - emerge from first principles of muscle contractile mechanics. Further, mechanical interactions of the muscle spindle with muscle-tendon dynamics reveal how motor commands to the muscle (alpha drive) versus muscle spindle (gamma drive) can cause highly variable and complex activity during active muscle contraction and muscle stretch that defy simple explanation. Depending on the neuromechanical conditions, the muscle spindle model output appears to 'encode' aspects of muscle force, yank, length, stiffness, velocity, and/or acceleration, providing an extendable, multiscale, biophysical framework for understanding and predicting proprioceptive sensory signals in health and disease.

Sarcolipin Exhibits Abundant RNA Transcription and Minimal Protein Expression in Horse Gluteal Muscle.

Ca2+ regulation in equine muscle is important for horse performance, yet little is known about this species-specific regulation. We reported recently that horse encode unique gene and protein sequences for the sarcoplasmic reticulum (SR) Ca2+-transporting ATPase (SERCA) and the regulatory subunit sarcolipin (SLN). Here we quantified gene transcription and protein expression of SERCA and its inhibitory peptides in horse gluteus, as compared to commonly-studied rabbit skeletal muscle. RNA sequencing and protein immunoblotting determined that horse gluteus expresses the ATP2A1 gene (SERCA1) as the predominant SR Ca2+-ATPase isoform and the SLN gene as the most-abundant SERCA inhibitory peptide, as also found in rabbit skeletal muscle. Equine muscle expresses an insignificant level of phospholamban (PLN), another key SERCA inhibitory peptide expressed commonly in a variety of mammalian striated muscles. Surprisingly in horse, the RNA transcript ratio of SLN-to-ATP2A1 is an order of magnitude higher than in rabbit, while the corresponding protein expression ratio is an order of magnitude lower than in rabbit. Thus, SLN is not efficiently translated or maintained as a stable protein in horse muscle, suggesting a non-coding role for supra-abundant SLN mRNA. We propose that the lack of SLN and PLN inhibition of SERCA activity in equine muscle is an evolutionary adaptation that potentiates Ca2+ cycling and muscle contractility in a prey species domestically selected for speed.

Evolution of Flight Muscle Contractility and Energetic Efficiency.

The powered flight of animals requires efficient and sustainable contractions of the wing muscles of various flying species. Despite their high degree of phylogenetic divergence, flight muscles in insects and vertebrates are striated muscles with similarly specialized sarcomeric structure and basic mechanisms of contraction and relaxation. Comparative studies examining flight muscles together with other striated muscles can provide valuable insights into the fundamental mechanisms of muscle contraction and energetic efficiency. Here, we conducted a literature review and data mining to investigate the independent emergence and evolution of flight muscles in insects, birds, and bats, and the likely molecular basis of their contractile features and energetic efficiency. Bird and bat flight muscles have different metabolic rates that reflect differences in energetic efficiencies while having similar contractile machinery that is under the selection of similar natural environments. The significantly lower efficiency of insect flight muscles along with minimized energy expenditure in Ca2+ handling is discussed as a potential mechanism to increase the efficiency of mammalian striated muscles. A better understanding of the molecular evolution of myofilament proteins in the context of physiological functions of invertebrate and vertebrate flight muscles can help explore novel approaches to enhance the performance and efficiency of skeletal and cardiac muscles for the improvement of human health.

The Structure, Function, and Physiology of the Fetal and Adult Acetylcholine Receptor in Muscle.

The neuromuscular junction (NMJ) is a highly developed synapse linking motor neuron activity with muscle contraction. A complex of molecular cascades together with the specialized NMJ architecture ensures that each action potential arriving at the motor nerve terminal is translated into an action potential in the muscle fiber. The muscle-type nicotinic acetylcholine receptor (AChR) is a key molecular component located at the postsynaptic muscle membrane responsible for the generation of the endplate potential (EPP), which usually exceeds the threshold potential necessary to activate voltage-gated sodium channels and triggers a muscle action potential. Two AChR isoforms are found in mammalian muscle. The fetal isoform is present in prenatal stages and is involved in the development of the neuromuscular system whereas the adult isoform prevails thereafter, except after denervation when the fetal form is re-expressed throughout the muscle. This review will summarize the structural and functional differences between the two isoforms and outline congenital and autoimmune myasthenic syndromes that involve the isoform specific AChR subunits.

НАУКОВО-ПЕДАГОГІЧНА ДІЯЛЬНІСТЬ КОЛЕСНИКОВА В. В.

Колесников Володимир Васильович – заслужений діяч науки, доктор біологічних наук, професор. Працював завідувачем кафедри гістології і очолював факультет ветеринарної медицини Одеського сільськогосподарського інституту. Він першим обґрунтував існування підтазового відділу черевної порожнини у деяких представників гризунів і комахоїдних, а також встановив етапи становлення придаткових зовнішніх статевих органів ссавців. В працях по морфології легень науковцем по-новому висвітлена функція бронхіальних шляхів і формування респіраторних відділів на прикладі дихальної системи великої рогатої худоби та коней.