PRLR x Tubulin

Hyperprolactinemia is prevalent in up to 16% of infertile males. Although the prolactin receptor (PRLR) is present on various testicular cells, the physiological role of this receptor in spermatogenesis remains elusive. The aim of this study is to delineate prolactin actions in rat testicular tissue. Serum prolactin, developmental expression of PRLR, signaling pathways associated, and gene transcription regulation in the testes were investigated. Serum prolactin and testicular PRLR expression was found to be significantly increased at pubertal and adult ages as compared to prepubertal. Further, PRLR activated the JAK2/STAT5 pathway, but not the MAPK/ERK and PI3K/AKT pathway in the testicular cells. Gene expression profiling following prolactin treatment in seminiferous tubule culture resulted in a total of 692 differentially expressed genes, of which 405 were upregulated and 287 were downregulated. Enrichment map analysis showed that prolactin target genes are involved in processes such as cell cycle, male reproduction, chromatin remodeling, and cytoskeletal organization. Novel gene targets of prolactin whose role in testes is unexplored were obtained and validated by qPCR. Additionally, 10 genes involved in cell cycle process were also validated; 6 genes (Ccna1, Ccnb1, Ccnb2, Cdc25a, Cdc27, Plk1) were found to be significantly upregulated, whereas 4 genes (Ccar2, Nudc, Tuba1c, Tubb2a) were found to be significantly downregulated in testes after treatment with prolactin. Taken together, the findings from this study suggest a crucial role of prolactin in male reproduction and identified target genes regulated by prolactin in the testes.

In addition to its established lactational roles, prolactin acts on multiple target tissues and its circulating levels are responsive to a range of physiological stimuli. The present study used immunohistochemistry to demonstrate that systemic administration of prolactin activates target cells in the arcuate nucleus and median eminence of the male mouse. Prolactin receptor stimulation results in the phosphorylation and thus activation of the signal transducer and activator of transcription (STAT)5 pathway. Interestingly, although, in the arcuate nucleus, this response was localised to cell nuclei, the median eminence displayed both nuclear and diffuse, non‐nuclear, phospho‐STAT5 (pSTAT5) staining. Dual‐label immunostaining demonstrated that, although the majority of nuclear pSTAT5 within the median eminence was located within vimentin‐positive tanycytes, the non‐nuclear staining occurred primarily in neuronal (βIII tubulin immunoreactive) elements. This conclusion was supported by the marked reduction of this signal in prolactin‐treated mice lacking neuronal prolactin receptors. A smaller reduction was also seen in animals lacking prolactin receptors on GABAergic but not glutamatergic neurones. These findings identify a new prolactin target tissue and, in doing so, support the proposal that the median eminence has a sensory role in addition to its established secretory function. The physiological significance of this prolactin response is unknown, although its rapidity (maximum within 2 minutes of i.p. injection) suggests that it may enable the early detection of an increase in circulating prolactin. It is also possibile that non‐nuclear prolactin‐generated pSTAT5 in the median eminence may have a local, non‐transcriptional, action. To this end, we used Evans Blue dye to demonstrate that elevated prolactin appears to reduce median eminence permeability and also that this effect is lost in animals lacking neuronal prolactin receptors.

Thyroid diseases have deleterious effects on lactation, litter growth and survival, and hinder the suckling-induced hormone release, leading in the case of hyperthyroidism, to premature mammary involution. To determine the effects of hypothyroidism (HypoT) on late lactation, we analyzed the effect of chronic 6-propyl-2-thiouracil (PTU)-induced HypoT on mammary histology and the expression of members of the JAK/STAT/SOCS signaling pathway, milk proteins, prolactin (PRLR), estrogen (ER), progesterone (PR) and thyroid hormone (TR) receptors, markers of involution (such as stat3, lif, bcl2, BAX and PARP) on lactation (L) day 21. HypoT mothers showed increased histological markers of involution compared with control rats, such as adipose/epithelial ratio, inactive alveoli, picnotic nuclei and numerous detached apoptotic cells within the alveolar lumina. We also found decreased PRLR, β-casein and α-lactoalbumin mRNAs, but increased SOCS1, SOCS3, STAT3 and LIF mRNAs, suggesting a decrease in PRL signaling and induction of involution markers. Furthermore, Caspase-3 and 8 and PARP labeled cells and the expression of structural proteins such as β-Actin, α-Tubulin and Lamin B were increased, indicating the activation of apoptotic pathways and tissue remodelation. HypoT also increased PRA (mRNA and protein) and erβ and decreased erα mRNAs, and increased strongly TRα1, TRβ1, PRA and ERα protein levels. These results show that lactating HypoT rats have premature mammary involution, most probably induced by the inhibition of prolactin signaling along with the activation of the LIF-STAT3 pathway.