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    • Recently higher grade varicoceles have been reported to be

    2021-09-14

    Recently, higher-grade varicoceles have been reported to be associated with higher nocturia levels [20], and we observed an interesting phenomenon in which the urine volume/water intake ratio dramatically increased in the rat model of experimental left varicocele (ELV); the decreases in hypothalamic VP levels occurred along with decreases in the GnRH level in this classic animal model of reproductive dysfunction. To confirm whether VP directly affects hypothalamic GnRH neurons, the distributions of GnRH and VP in the hypothalamus were examined, and the possible interaction between GnRH and VP was explored by NPS-2143 microscopic immunohistochemistry and an in situ proximity ligation assay (PLA). Furthermore, serial cytological experiments were conducted with an immortalized GnRH-expressing cell line, GT1-7, to verify the effects of VP on GnRH neurons. The results of the present study provide a preliminary reference for the exploration of the direct effect of VP on GnRH neurons, which may be benefit studies on reproductive regulation.
    Experimental procedure
    Results
    Discussion Due to the vital role played by GnRH neurons in the hypothalamic-pituitary-gonadal axis, the definite effects of VP on GnRH secretion have gained widespread attention. Based on the interesting phenomenon that the urine volume/water intake ratio dramatically increased but the blood pressure levels decreased in experimental varicocele rats, this study reported the characteristics of hypothalamic GnRH and VP expression, found histomorphological clues of a direct correlation between VP and GnRH neurons, and detected the effects of intracellular and extracellular VP on GnRH synthesis and secretion in vitro. The results of the present study provide a preliminary reference for the direct effect of VP on GnRH neurons and suggest that extracellular VP can effectively promote GnRH synthesis. The ELV rat model is a classic reproductive injury model mimicking the pathology of male varicocele. Our previous study indicated that ELV establishment significantly attenuated the hypothalamic GnRH expression and spine density in the Arc, implying that the changes in GnRH neurons participate in ELV progression [24]. Additionally, the ratio of urine output to water intake was calculated in the present study to equilibrate the effects of various levels of water intake on urine output, although increased water intake directly leads to the dilution of urine and an increase in urine output. VP, also known as antidiuretic hormone (ADH), is essential for water regulation and has negative effects on blood pressure [25]. The higher urine volume/water intake ratio and the lower blood pressure levels in ELV rats made VP become a focus of research. The decreases in the fluorescence intensities of immunoreactive substances of both GnRH and VP in the Arc and the ME plane of the hypothalamus in ELV rats were restored to normal levels by ELV repair, indicating that the relationship between hypothalamic GnRH and VP is one of co-variation. For ArcL adjacent to ME is the key nucleus where GnRH neurons scatters, we supposed VP projected to ME have some effects on GnRH synthesis and secretion through extracellular or intracellular pathway. It has been reported in the literature that VP may promote the secretion of GnRH by activating the Kiss1-GPR54 system [26,27]. However, it is not yet known whether VP is directly related to GnRH neurons and GnRH secretion. Immunofluorescence histochemical staining of hypothalamic sections revealed that GnRH and VP were distributed throughout the ArcL and ME and were localized in a somewhat overlapping fashion. The results showed the expressions of GnRH and VP, as well as the colocalization sites, decreased after ELV and restored by ELV repair. The covariation and colocalization of GnRH and VP spark speculation about the interactions between them two. To further verify the existence of interactions between VP and GnRH, in situ PLA, and electron microscopic immunohistochemistry were employed to examine whether there is a closely adjacent relationship or a coexistent relationship that has been deemed as the basis of interactions between hypothalamic VP and GnRH. In situ PLA, a straightforward process for identifying protein interactions in their natural context that has rarely been used in hypothalamic sections [28], was used to observe the co-localization region, ArcL, of GnRH and VP. PLA-positive signals were found in the cytoplasm and neurites of neurons in the ArcL, consistent with the electron microscopic immunohistochemistry results revealing immunoreactive substances of GnRH and VP in the same and adjacent axon terminals. The proximity of protein locations is a prerequisite for protein-protein interactions; therefore, experiments were conducted in GT1-7 cells to investigate whether AVP influences the synthesis and release of GnRH.