Martínez Hernández,  Jesús

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Martínez Hernández, Jesús

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Martínez Hernández, Jesús

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Biología Celular e Histología

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Open Access
Redox profiling of preovulatory follicular fluid in the donkey is species-specific, and contributes to modulate sperm function
(Nature Research, 2025-02-23) Catalán, Jaime; Padilla, Lorena; Maside, Carolina; Yánez-Ortiz, Iván; Tvarijonaviciute, Asta; Barranco Cascales, Isabel; Bonet, Sergi; Miró, Jordi; Yeste, Marc; Martínez Hernández, Jesús; Medicina y Cirugía Animal; Facultad de Veterinaria
The follicular fluid (FF) is crucial for providing oocytes with an ideal environment that promotes their development and maturation. Not only does this fluid supply nutrients and hormones, but also other components that protect both follicular cells and the oocyte itself from potential harmful factors, such as those inducing oxidative stress (OS). The FF has also been suggested to have beneficial effects on sperm when they reach the oviduct. The objectives of this study were to describe the presence and activity levels of redox biomarkers in the preovulatory follicular fluid (PFF), and to analyze the impact of PFF on the motility, viability and reactive oxygen species (ROS) levels of donkey sperm. For this purpose, ten PFF samples obtained from Catalan jennies and nine ejaculates collected from Catalan donkeys were used. Redox biomarkers -including enzymatic and non-enzymatic antioxidants, and oxidative biomarkers- were analyzed in jennies' PFF. After collection, each semen sample was split into two aliquots of equal volume. The two aliquots were centrifuged to remove the seminal plasma; one pellet was resuspended in Tris Buffered Medium (TBM), and the other was resuspended in TBM supplemented with 20% PFF. Motility parameters (CASA) and other semen quality biomarkers (flow cytometry) were assessed after 0, 60 and 120 min of incubation at 38 °C. Exposure of donkey sperm to PFF reduced intracellular ROS levels and helped maintain sperm motility and viability. These findings suggest that, in the oviduct, the PFF components protect donkey sperm from oxidative stress. Furthermore, knowing better the composition of donkey PFF in terms of antioxidant biomarkers may be used to improve the formulation of media for oocyte maturation and fertilization in this and other species.
Open Access
Histología, actividad proliferativa y apoptótica durante la recrudescencia testicular tras fotoperiodo corto en el epitelio seminífero del hámster sirio (Mesocricetus auratus)
(Universidad de Murcia, 2020-12-04) Martínez Hernández, Jesús; Ferrer Cazorla, Concepción; Pastor García, Luis Miguel; Escuela Internacional de Doctorado
Numerosos mamíferos presentan una reproducción de carácter estacional que hace que las crías nazcan en el mejor momento para su supervivencia. Los machos padecen una infertilidad temporal adquirida tras un proceso de regresión testicular del cual tienen que recuperarse, en un proceso conocido como recrudescencia, en el que los testículos vuelven a producir espermatozoides competentes para la reproducción. El objetivo de esta tesis doctoral ha sido estudiar el túbulo seminífero del hámster sirio durante la recrudescencia espontánea tras la exposición a un fotoperiodo corto. Para ello, se han determinado sus cambios histomorfométricos así como las actividades de proliferación y apoptosis en las células germinales y en la célula de Sertoli durante este proceso. Se utilizaron 53 hámsteres sirios machos mantenidos en fotoperiodo de 14:10h luz-oscuridad. De ellos, 5 se utilizaron como grupo de Control mantenidos en el mismo fotoperiodo (14:10h luz-oscuridad). Los otros 48 animales se sometieron a un fotoperiodo de 8:16h de luz-oscuridad. Se establecieron los siguientes grupos de estudio: regresión media (MReg), regresión fuerte (SReg), regresión total (TReg), recrudescencia inicial (IR), recrudescencia avanzada (AR) y recrudescencia total (TR). Las siguientes técnicas histológicas fueron utilizadas: hematoxilina-eosina, inmunohistoquímica de vimentina y PCNA, histoquímica de lectinas y TUNEL, y, para fluorescencia: doble inmunofluorescencia de vimentina y PCNA. Se realizó también un estudio morfométrico. Durante la recrudescencia hay una recuperación gradual del volumen testicular, tubular y del epitelio seminífero. El volumen intersticial, la longitud y el diámetro tubular aumentaron principalmente en la primera mitad del proceso. La actividad proliferativa de las espermatogonias fue siempre mayor que la del grupo Control y la actividad apoptótica disminuyó en la primera mitad de la recrudescencia. Las lectinas mostraron cambios en el patrón de glucoconjugados al inicio del proceso, aunque gradualmente se fue recuperando un patrón similar al del grupo Control. Por otro lado, los espermatocitos y espermátidas en apoptosis mostraron gran afinidad por las lectinas PNA, GNA, AAA y Con-A, además, para los espermatocitos en apoptosis, también la lectina LTA. En todos los grupos se observó células de Sertoli vimentina+/TUNEL + y células vimentina+/PCNA+. El índice de apoptosis para la célula de Sertoli fue mayor en MRg y SRg que en el resto de los grupos mientras que su índice de proliferación fue mayor en TRg e IR respecto al resto de grupos. El número total de células de Sertoli aumentó entre TRg, IR y AR, donde alcanzó valores del grupo Control. En conclusión, la recrudescencia espontánea del testículo de hámster sirio comprende dos fases histomorfométricas. La primera con incremento del diámetro y longitud tubular, con aumento del volumen intersticial y, una segunda, con incremento gradual de diámetro tubular. La recuperación del epitelio seminífero se debe al aumento de proliferación en las espermatogonias en la recrudescencia a la par que la apoptosis de las células germinales disminuye a valores del control a la mitad del proceso. La histoquímica de lectinas confirmó su utilidad para detectar células germinales en apoptosis. Finalmente, el aumento de proliferación y diminución de apoptosis de la célula de Sertoli permitió restablecer su número después de la pérdida ocurrida durante la regresión, hecho fundamental para la recuperación de la fertilidad. La constatación de una tasa de recambio (proliferación / apoptosis) en los animales mantenidos en fotoperiodo largo indican que la célula de Sertoli no está terminalmente diferenciada en el hámster sirio adulto.
Open Access
Extracellular vesicles would be involved in the release and delivery of seminal TGF-β isoforms in pigs
(Frontiers Media, 2023-02-10) Padilla, Lorena; Barranco, Isabel; Parra, Ana; Parrilla, Inmaculada; Rodríguez Martínez, Heriberto; Lucas, Xiomara; Roca, Jordi; Martínez Hernández, Jesús; Pastor García, Luis Miguel; Medicina y Cirugía Animal
Introduction: pig seminal plasma (SP) is rich in active forms of all three isoforms (1-3) of transforming growth factor β (TGF-β), a chemokine modulatory of the immune environment in the female genital tract once semen is delivered during mating or artificial insemination (AI). The present study aimed to examine how TGF-βs are secreted by the epithelium of the male reproductive tract and how they are transported in semen, emphasizing the interplay with seminal extracellular vesicles (sEVs). Methods: Source of TGF-βs was examined by immunohistochemistry in testis, epididymis, and accessory sex glands, by immunocytochemistry in ejaculated spermatozoa, and by Luminex xMAP® technology in SP and sEVs retrieved from healthy, fertile male pigs used as breeders in AI programs. Results: All three TGF-β isoforms were expressed in all reproductive tissues explored and would be released into ductal lumen either in soluble form or associated with sEVs. Ejaculated spermatozoa expressed all three TGF-β isoforms, both inside and outside, probably the outer one associated with membrane-bound sEVs. The results confirmed that pig SP contains all three TGF-β isoforms and demonstrated that a substantial portion of them is associated with sEVs. Discussion: Seminal EVs would be involved in the cellular secretion of the active forms of seminal TGF-β isoforms and in their safe transport from the male to the female reproductive tract.
Open Access
Heterogeneity of mesenchymal cells in human amniotic membrane at term
(Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2024) Cortés Sandoval, Salvador; Serrano Sánchez, Mª Isabel; Ferrer, Concepción; Delgado, Juan L.; Insausti, Carmen L.; Blanquer, Miguel; Beltrán Frutos, Ester; Martínez Hernández, Jesús; Pastor García, Luis Miguel; Seco Rovira, Vicente
There is increasing interest in understanding the tissue biology of human amniotic membrane (hAM) given its applications in medicine. One cellular component is mesenchymal cells, which can be extracted, cultured and differentiated "in vitro" into various cell types. These studies show that there is heterogeneity among mesenchymal cells. The aim of this work is to study the membrane "in situ" to determine whether this cellular heterogeneity exists. The hAMs were obtained from caesarean deliveries at term and analyzed by histological techniques. Types I-III mesenchymal cells and Hofbauer were distinguished by light microscopy. Histochemically, mesenchymal cell types showed successively increasing positivity to: PAS, vimentin, fibronectin, and Concanavalin-A; VGEF, TGF-β2, PDGF-C, FGF-2. By the semiquantitative point of view, the percentage of Type II cells was 60%, significantly higher than the other types. With transmission electron microscopy, an intermediate cell type between II-III was observed. Strong vesiculation of the rough endoplasmic reticulum (RER) with exocytosis was observed. In addition, an accumulation of a similar material to the extracellular matrix in the RER caused its dilation especially in type IIITEM cells. Some of this material acquired a globular structure. These structures were also found free in the extracellular matrix. In conclusion, the mesenchymal cells of the fibroblastic layer of the hAMs studied are heterogeneous, with some undifferentiated and others with a probably senescent fibroblastic phenotype with accumulation in their RER of fibronectin. These results may be of interest to extract mesenchymal cells from hAMs for use in regenerative medicine and to better understand the mechanisms of fetal membrane rupture
Open Access
The oxytocin receptor in spermatozoa may originate from both spermatogenesis and epididymal maturation, and regulates capacitation
(Wiley, 2025-09-27) Garriga, Ferran; Ahmad, Adeel; Padilla, Lorena; Maside, Carolina; Bonet, Sergi; Barranco Cascales, Isabel; Roca, Jordi; Pastor García, Luis Miguel; Yeste, Marc; Martínez Hernández, Jesús; Medicina y Cirugía Animal; Facultad de Veterinaria
Background: The oxytocin receptor (OR) is a G-protein-coupled receptor recently identified in human spermatozoa, whose origin and role in sperm physiology remain unknown. Objectives: In this study, using the pig as a model, we examine the presence of the OR in ejaculated spermatozoa through immunofluorescence and immunoblotting, and investigate the receptor's origin in the male gamete via immunohistochemistry in testicular and epididymal tissues. Additionally, we assess the involvement of the OR in in vitro capacitation and the acrosome reaction by utilizing physiological concentrations of agonists (oxytocin and carbetocin) and an antagonist (L-371,257). Results: The results indicate that, in addition to the expected presence in ejaculated spermatozoa, the OR is expressed during spermatogenesis. Besides, this receptor is found in Leydig and Sertoli cells, as well as in the principal, basal, and apical cells of the epididymis. Furthermore, our data suggest that, during epididymal maturation, the OR could be incorporated in spermatozoa via extracellular vesicles within the apical blebs. The OR is involved in sperm capacitation, as the combination of the antagonist (L-371,257) and the agonist (carbetocin) increases intracellular calcium levels and membrane lipid disorders, which are known as capacitation markers. Conclusions: The presence of the OR in mammalian spermatozoa could originate from both spermatogenesis and epididymal maturation. Moreover, in the male gamete, this receptor regulates sperm capacitation by interacting with its ligand in the female reproductive tract.
Open Access
Testicular histomorphometry and the proliferative and apoptotic activities of the seminiferous epithelium in Syrian hamster during spontaneous recrudescence after exposure to short photoperiod
(© 2018 Blackwell Verlag GmbH., 2018-10-01) Ferrer Cazorla, Concepción; Canteras, Manuel; Sánchez-Huertas, María del Mar; Beltrán Frutos, Ester; Martínez Hernández, Jesús; Pastor García, Luis Miguel; Seco Rovira, Vicente; Biología Celular e Histología
Syrian hamsters are photoperiodic rodents in which reproduction, including testicular function, is stimulated by long photoperiod exposure and curtailed by exposure to a short photoperiod. The objectives of this study were to characterize the testis histomorphometrically and to determine the role of the proliferation and apoptosis phenomena in the recovery of the seminiferous epithelium during spontaneous recrudescence after exposure to short photoperiod. The study was performed using conventional light microscopy, proliferating cell nuclear antigen and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP in situ nick end labelling staining, image analysis software, and transmission electron microscopy in three recrudescence groups: initial recrudescence (IR), advanced recrudescence (AR), and total recrudescence (TR). Morphometrically, the results pointed to the gradual recovery of the testicular and tubular volumes, as well as of the seminiferous epithelium. Among the IR and AR groups, the increase in testicular and tubular volumes was accompanied by an increase in tubular diameter and length, with an increase in interstitial volume. From AR to TR, there was an increase in the tubular and total volumes, but, in this case, with a gradual increase in tubular diameter. Recovery of the seminiferous epithelium was accompanied by changes in apoptosis and proliferation activities. The first decreased half way through the process and the second remained higher than the control levels throughout the recrudescence stage. Ultrastructurally, alterations in the spermatozoa were observed, which indicated that spermiogenesis was not yet completely normal. In conclusion, spontaneous testicular recrudescence in Syrian hamster comprises two histomorphometrical phases, the first related to an increase in tubular length and diameter and interstitial volume, and the second depending principally on the gradual increase in tubular diameter. The restoration of the seminiferous epithelium is due to apoptosis reaching normal values in the AR group accompanied by higher proliferative activity than that observed in the Control group.
Open Access
Granulocyte-macrophage colony stimulating factor (GM-CSF) is fully expressed in the genital tract, seminal plasma and spermatozoa of male pigs
(Nature Research, 2020-08-07) Padilla, Lorena; Barranco, Isabel; Lucas, Xiomara; Rodríguez-Martínez, Heriberto; Roca, Jordi; Parrilla, Inmaculada; Martínez Hernández, Jesús; Pastor García, Luis Miguel; Medicina y Cirugía Animal
Granulocyte-macrophage colony stimulating factor (GM-CSF) is a pro-inflammatory cytokine identified in boar seminal plasma (SP) but until now unexplored in terms of place of production and its association to spermatozoa. This study aimed to explore these aspects by evaluating the presence of GM-CSF in porcine reproductive organs (testes, epididymis and accessory sex glands), SP and mature spermatozoa (from cauda epididymis and ejaculated) using Western blot (WB), immunohistochemistry and immunocytochemistry. Positive labelling was obtained in tissues, SP and spermatozoa. In reproductive organs, WB revealed three forms of GM-CSF with different glycosylation degrees (15, 31 and 40 kDa). In SP and epididymal fluid, the GM-CSF appeared only in its active form while in spermatozoa the GM-CSF form present varied among sperm sources. Non-viable spermatozoa showed more GM-CSF than viable spermatozoa (14.87 ± 1.98 RU vs. 7.25 ± 0.52 RU) of fluorescence intensity. In conclusion, GM-CSF is widely present in the reproductive tract of male pigs, attached to the spermatozoa already in the epididymis as well as verted to SP. Consequently, the GM-CSF ought to regulate male genital tract and sperm function as well as mediating initial inflammatory responses and further mediating later immune actions by the female to semen deposition.
Open Access
HSP47 expression in the hamster Sertoli cell: An immunohistochemical study
(Universidad de Murcia. Departamento de Biología Celular e Histología, 2024) Serrano-Sánchez, María Isabel; Ferrer, Concepción; Beltrán Frutos, Ester; Martínez Hernández, Jesús; Pastor García, Luis Miguel; Seco Rovira, Vicente
HSP47, a chaperone whose main function is the maturation of collagen molecules, is considered a marker of fibrotic diseases. Increased collagen synthesis in the testis has been associated with various pathologies leading to seminiferous tubule regression. Our aim was to study whether HSP47 is expressed in hamster Sertoli cells both in the adult and in two physiological situations of seminiferous tubule atrophy: irreversible testicular ageing and testicular regression due to short photoperiod (reversible). Eighteen animals were divided as follows: a group of 6 young animals aged 6 months, a group of 6 animals aged 24 months, which were exposed to a long photoperiod, and a final group of 6 young animals subjected to a short photoperiod. Testicular samples were fixed in methacarn and an immuno-histochemical technique was used to detect HSP47. A semiquantitative study of this protein expression was performed between tubular sections of aged animals with complete spermatogenesis and arrested spermatogenesis and tubular sections with arrest spermatogenesis of photoinhibited testes. Sertoli cells were positive for HSP47, the intensity being greater in tubular sections with arrested spermatogenesis in both aged and photoinhibited animals. Semiquantitative analysis corroborated this observation in the sense that the expression of this protein differed according to the functional state of the seminiferous tubules. Thus, the ratio of immunoreactivity was significantly higher in tubular sections with arrested spermatogenesis in aged animals compared with regressed animals, and in the latter compared with those whose tubular sections showed complete spermatogenesis. In conclusion, HSP47 expression in Sertoli cells was found for the first time in mammals. Moreover, increased expression seemed to be related to the degree of seminiferous atrophy epithelium and to the reversible or nonreversible physiological state of this epithelium.
Open Access
Lectin binding pattern of glycoconjugates during spontaneous testicular recrudescence in Syrian hamster (Mesocricetus auratus) after exposure to short photoperiod
(Wiley, 2019-02-01) Ferrer Cazorla, Concepción; Serrano-Sánchez, María Isabel; Beltrán Frutos, Ester; Martínez Hernández, Jesús; Pastor García, Luis Miguel; Seco Rovira, Vicente; Biología Celular e Histología
Lectin histochemistry was used to characterize glycoconjugates and cellular apoptosis in the seminiferous epithelium and interstitium of hamster testis during spontaneous recrudescence. An increase in the LTA lectin affinity was observed in spermatids in the Golgi phase. An increase in labelling of PNA and Con-A lectin in acrosome of spermatids (acrosome phase) as well increased labelling with Con-A in spermatids (cap phase) was observed. Spermatocytes showed decreased affinity with PNA and AAA lectins and an increase in positivity for LTA and GNA lectins. Spermatogonia showed a slight decrease in positivity to WGA and an increase in labelling with Con-A, and a decreased affinity for the AAA lectin. At the end of recrudescence all these germinal cells showed a similar pattern to the control. The Sertoli cells showed a gradual decrease in labelling with the GNA lectin and the Leydig cells an increase in labelling with Con-A and GNA. Particularly unusual was the observation of apoptotic spermatocytes and spermatids positive for PNA, GNA, AAA and Con-A, together with spermatocytes positive to LTA. In conclusion the normal lectin pattern is recovered during testis recrudescence and germ cell apoptotic activity is low, as is observed by specific lectins for germ cells in apoptosis.