Sperm Chromatin Status and DNA Fragmentation in Mouse Species with Divergent Mating Systems.

Sperm DNA integrity and chromatin status serve as pivotal indicators of sperm quality, given their intricate link to sperm function, embryo development, and overall fertility. Defects in chromatin compaction, which are often associated with compromised protamine content, can lead to damaged DNA strands. In this study, the chromatin status and possible correlation with DNA damage was assessed in males of three mouse species: Mus musculus, M. spretus, and M. spicilegus. We employed various staining methods, including aniline blue, methylene blue (Diff-Quik), toluidine blue, and chromomycin A3, to assess chromatin compaction in cauda epididymal sperm. Samples were also analyzed by the sperm chromatin structure assay (SCSA) to estimate DNA fragmentation (%tDFI, %HDS). Analyses were carried out on freshly collected sperm and cells incubated for 3 h in a HEPES-buffered modified Tyrode's medium simulating conditions of the female reproductive tract. Notably, the analysis of chromatin status yielded minimal abnormal values across all three species employing diverse methodologies. SCSA analyses revealed distinct variations in %tDFI between species. Following sperm incubation, the percentages of sperm stained with methylene blue exhibited differences among the species and were significantly correlated to the DNA fragmentation index. HDS demonstrated correlations with the percentages of sperm stained by aniline blue, methylene blue, and chromomycin A3. Overall, chromatin compaction was high across all species, with limited differences among them. The relationship between chromatin status and DNA integrity appeared to be related to levels of sperm competition among species.

SPAG17 mediates nuclear translocation of protamines during spermiogenesis.

Protamines (PRM1 and PRM2) are small, arginine-rich, nuclear proteins that replace histones in the final stages of spermiogenesis, ensuring chromatin compaction and nuclear remodeling. Defects in protamination lead to increased DNA fragmentation and reduced male fertility. Since efficient sperm production requires the translocation of protamines from the cytoplasm to the nucleus, we investigated whether SPAG17, a protein crucial for intracellular protein trafficking during spermiogenesis, participates in protamine transport. Initially, we assessed the protein-protein interaction between SPAG17 and protamines using proximity ligation assays, revealing a significant interaction originating in the cytoplasm and persisting within the nucleus. Subsequently, immunoprecipitation and mass spectrometry (IP/MS) assays validated this initial observation. Sperm and spermatids from Spag17 knockout mice exhibited abnormal protamination, as revealed by chromomycin A3 staining, suggesting defects in protamine content. However, no differences were observed in the expression of Prm1 and Prm2 mRNA or in protein levels between testes of wild-type and Spag17 knockout mice. Conversely, immunofluorescence studies conducted on isolated mouse spermatids unveiled reduced nuclear/cytoplasm ratios of protamines in Spag17 knockout spermatids compared to wild-type controls, implying transport defects of protamines into the spermatid nucleus. In alignment with these findings, in vitro experiments involving somatic cells, including mouse embryonic fibroblasts, exhibited compromised nuclear translocation of PRM1 and PRM2 in the absence of SPAG17. Collectively, our results present compelling evidence that SPAG17 facilitates the transport of protamines from the cytoplasm to the nucleus.

Adaptive functional specialisation of architectural design and fibre type characteristics in agonist shoulder flexor muscles of the llama, Lama glama.

Like other camelids, llamas (Lama glama) have the natural ability to pace (moving ipsilateral limbs in near synchronicity). But unlike the Old World camelids (bactrian and dromedary camels), they are well adapted for pacing at slower or moderate speeds in high-altitude habitats, having been described as good climbers and used as pack animals for centuries. In order to gain insight into skeletal muscle design and to ascertain its relationship with the llama's characteristic locomotor behaviour, this study examined the correspondence between architecture and fibre types in two agonist muscles involved in shoulder flexion (M. teres major - TM and M. deltoideus, pars scapularis - DS and pars acromialis - DA). Architectural properties were found to be correlated with fibre-type characteristics both in DS (long fibres, low pinnation angle, fast-glycolytic fibre phenotype with abundant IIB fibres, small fibre size, reduced number of capillaries per fibre and low oxidative capacity) and in DA (short fibres, high pinnation angle, slow-oxidative fibre phenotype with numerous type I fibres, very sparse IIB fibres, and larger fibre size, abundant capillaries and high oxidative capacity). This correlation suggests a clear division of labour within the M. deltoideus of the llama, DS being involved in rapid flexion of the shoulder joint during the swing phase of the gait, and DA in joint stabilisation during the stance phase. However, the architectural design of the TM muscle (longer fibres and lower fibre pinnation angle) was not strictly matched with its fibre-type characteristics (very similar to those of the postural DA muscle). This unusual design suggests a dual function of the TM muscle both in active flexion of the shoulder and in passive support of the limb during the stance phase, pulling the forelimb to the trunk. This functional specialisation seems to be well suited to a quadruped species that needs to increase ipsilateral stability of the limb during the support phase of the pacing gait. Compared with other species, llama skeletal muscles are well suited for greater force generation combined with higher fatigue resistance during exercise. These characteristics are interpreted as being of high adaptive value, given the llama's habitat and its use as a pack animal.

Muscular partitioning in the semitendinosus muscle of the pig / Compartimientos musculares del músculo semitendinoso del cerdo

Neuromuscular compartments are subvolumens supplied by a primary nerve branch with homogeneous fiber characteristics. This muscular organization is important for understanding the muscle function in relation to postural and dynamic implications and also is relevant for appraising meat quality. Current researches associate fiber muscle characteristics with perimortem processes that regulate muscle transformation into meat. It is known that pig muscle has four heavy chain myosin isoforms: I, IIa, IIx and IIb. Castrated male pigs were slaughtered at 100 kg live weight. Semitendinosus muscle samples of each subvolumen determined previously (R1, R2, R3, R4) were frozen in liquid nitrogen and reacted for myofibrillar ATPase and NADH-TR for determining fiber types and oxidative capacity respectively. It were identified I, IIA and IIX/IIB fiber types. The frequency distribution of the I, IIA, and IIX/IIB fiber types were significantly greater in R1- R4, R4 and R3 respectively. The CSA of all fiber types was significantly larger in R1 than other compartments, notwithstanding CSA did not vary significantly among fiber types; oxidative capacity was significantly greater in R4. These results show a heterogeneousness of the muscle which would explain probably the disagreements documented between characteristics of fiber and meat quality traits.

Los compartimientos neuromusculares son subvolúmenes abastecidos por un ramo nervioso primario, los cuales poseen características fibrilares homogéneas. Esta organización muscular es importante para entender la función muscular en relación tanto en la estática y dinámica como en la apreciación de la calidad de la carne. Actuales investigaciones asocian características fibrilares con procesos perimorten que regulan la transformación del músculo en carne. Es conocido que en el músculo del cerdo están presentes cuatro cadenas pesadas de miosina: I, IIa, IIx y IIb. Cerdos machos castrados fueron faenados a los 100K kg de peso vivo. Muestras musculares de cada subvolumen del músculo semitendinoso, previamente determinados (R1, R2, R3, R4), fueron congeladas en nitrógeno líquido y tratadas con las reacciones de ATPasa miofribrilar y NADH-TR para determinar los tipos fibrilares y la capacidad oxidativa, respectivamente. Fueron identificadas los tipos fibrilares I, IIA y IIX/IIB. La frecuencia de distribución de los tipos fibrilares I, IIA, y IIX/IIB fueron significativamente mayores en R1- R4, R4 y R3, respectivamente. La CSA de todos los tipos fibrilares fueron significativamente mayores en R1 que en los otros compartimientos, sin embargo CSA no varía en forma significativa entre los tipos fibrilares; la capacidad oxidativa fue significativamente mayor en R4. Estos resultados evidencian una heterogeneidad muscular la cual podría probablemente explicar los desacuerdos documentados entre las características de las fibras y su relación con la evaluación de la calidad de la carne.

Distribution of the internal pudendal artery in male and female llama (Lama glama).

The aim of this research has been to describe the internal pudendal artery distribution in male and female llama and to compare it with that of other domestic animals including the one-humped camel (Camelus dromedarius). The arterial system was perfused with a solution of 14% coloured plaster and preserved in a solution of a 10% formalin, 3% carbolic acid and 3% glycerine. The systematic dissection was made using traditional working techniques and standard instruments. The internal pudendal artery is the ventral terminal branch of the internal iliac artery at the level of the third sacral vertebra. The main supply of the pelvic organs comes from the prostatic or vaginal arteries; notwithstanding these arteries arise from the internal pudendal artery, showing an important difference between ruminants and pig (long iliac type). Similarities between the distribution of the internal pudendal artery of the llama and those obtained in the camel provide strong evidence of a common phylogenetic origin.

Systematic study of the internal iliac artery in llama (Lama glama).

The aim of this research was to determine the origins of the parietal and visceral branches of the internal iliac artery of the llama and to match those with the known types and classifications in the human being and domestic animals. The internal iliac artery divides at the level of the third sacral vertebra into the caudal gluteal and internal pudendal arteries corresponding to an intermediate long iliac type. It gives off the following collateral branches: umbilical, cranial gluteal, obturator and iliolumbar arteries. The intrapelvic visceral branches, vaginal or prostatic arteries, arise from the internal pudendal artery as occurs in carnivores. This is an important difference in relation to the distribution in characteristic long iliac and artiodactyla types. The origins of the parietal and visceral branches of internal iliac artery in the llama have characteristics in common with the domestic cat, which belongs to the carnivores. In relation to the Adachi classification, the internal iliac artery of the llama will be included in the IVa type. We believe that this study is a contribution of a new aspect in teaching and research in comparative anatomy.

Fiber muscle types in adult female pigs as determined by combinating histochemical and immunohistochemical methods

Las fibras musculares esqueléticas del cerdo han sido tradicionalmente tipificadas como I, IIA y IIB, más sus formas híbridas, determinando la actividad de la enzima miosina ATPasa miofibrilar. Recientemente, la utilización de anticuerpos monoclonales contra isoformas de cadena pesada de miosina, electroforesis y estudios moleculares, han documentado la existencia, además, de la isoforma IIx. El objetivo de este estudio fue determinar la existencia de las isoformas de miosina, presentes en los distintos tipos fibrilares, utilizando técnics histoquímicas e inmunohistoquímicas combinadas, dentro de una unidad experimental compuesta por muestras musculares del M. longissimo del dorso, en cerdas de 150 kg promedio. Las muestras fueron obtenidas 45 minutos después de la faena, mediante escisión y congeladas en acetona enfriada con hielo seco. Cortes seriados de 10 µm de espesor fueron tratados por la técnicas de mATPasa modificada por Nwoye (1982), a pH 4,6, NADH-TR e inmunohistoquímica. Histoquímicamente fueron identificados cinco grupos de fibras: tipo I oscuras, IIA claras, y tres tipos intermedios. Los ensayos inmunohistoquímicos permitieron identificar las isoformas ß lenta I, IIa, IIx y IIb presentes en fibras de tipo I, IIA, IIX, IIB y un grupo híbrido IIAX