Regulating gonad inhibition and vitellogenin/vitellin induction in Penaeus monodon using mature GIH fusion protein and polyclonal antisera.

Gonad inhibiting hormone (GIH), type II class of the CHH family neuropeptides, is released by the neurohaemal XO-SG complex of the eyestalk. The inhibitory function of GIH has a pivotal role in gonad development and reproduction. In this study, we report the expression and production of a thioredoxin-fused mature GIH protein (mf-PmGIH) of Penaeus monodon in a bacterial system and its use as antigen to raise polyclonal antiserum (anti-mf-PmGIH). The mature GIH gene of 237bp that codes for 79 amino acids, was cloned into the Escherichia coli thioredoxin gene fusion expression system. The expression vector construct (mf-PmGIH+pEt32a+) upon induction produced 32.16kDa mature GIH fusion protein (mf-PmGIH)·The purified fusion protein was used as exogenous GIH and as antigen to raise polyclonal antisera. The fusion protein when injected into juvenile shrimp significantly reduced vitellogenin/vitellin levels by 31.55% within 72h in comparison to the controls showing the gonad inhibiting property. Vitellogenin/vitellin levels were significantly induced by 74.10% within 6h when polyclonal antiserum (anti-mf-PmGIH - 1:500) was injected in P. monodon. Anti-mf-PmGIH immunolocalized GIH producing neurosecretory cells in the eyestalk of P. monodon. The present manuscript reports an innovative means of gonad inhibition and vitellogenin/vitellin induction with thioredoxin fused GIH and antisera developed.

Ovarian regulation of neuromedin U and its local actions in the ovary, mediated through neuromedin U receptor 2.

Neuromedin U (NMU) was originally identified as an anorexigenic peptide that modulates appetite as well as energy homeostasis through the brain-gut axis. Although growing evidence has linked NMU activity with the development of female reproductive organs, no direct expression of and function for NMU in these organs has been pinpointed. Using a superovulated rat model, we found that NMU is directly expressed in the ovary, where its transcript level is tightly regulated by gonadotropins. Ovarian microdissection and immunohistochemical staining showed clearly that NMU is expressed mainly in theca/interstitial cells and to a moderate extent in granulosa cells. Primary cell studies together with reporter assays indicated the Nmu mRNA level in these cells is strongly induced via cAMP signaling, whereas this increase in expression can be reversed by the degradation message residing within its 3'-untranslated region, which recruits cis-acting mRNA degradation mechanisms, such as the gonadotropin-induced zinc finger RNA-binding protein Zfp36l1. This study also demonstrated that NMUR2, but not NMUR1, is the dominant NMU receptor in the ovary, where its expression is restricted to theca/interstitial cells. Treatment with NMU led to induction of the early response c-Fos gene, phosphorylation of extracellular signal-regulated kinase 1/2, and promotion of progesterone production in both developing and mature theca/interstitial cells. Taken as a whole, this study demonstrates that NMU and NMU receptor 2 compose a novel autocrine system in theca/interstitial cells in which the intensity of signaling is tightly controlled by gonadotropins.

Modulation of microvascular permeability in the preovulatory rat ovary by an ovulatory gonadotropin stimulus.

OBJECTIVE: To characterize the size selectivity of the rat ovarian vasculature and its changes after gonadotropin induction of ovulation. DESIGN: Experimental study. SETTING: Obstetrics and Gynecology Department. ANIMAL(S): Immature, female Sprague-Dawley rats. INTERVENTION(S): Rats were pretreated with equine chorionic gonadotropin, and ovaries were retrieved either 48 hours later or at any of several time points during ovulation induced by hCG. Fluorescein isothiocyanate-labeled Ficoll was injected 10 minutes before ovarian sampling, and the distribution of Ficoll was measured in plasma and ovarian extracts. MAIN OUTCOME MEASURE(S): The Ficoll data were analyzed according to a two-pore model to acquire information on small (rS) and large (rL) pore radii as well as the number of large pores reflected by the large pore fraction of the hydraulic conductance (LpS%) at each periovulatory time interval. RESULT(S): Before hCG, rS and rL were 54.7 ± 1.2 Å (mean ± SEM) and 149.3 ± 5.3 Å, respectively. At this preovulatory stage, LpS% was 7.1% ± 3.2%. Stimulation with hCG caused close to a three-fold increase in LpS% at 2 and 4 hours (20.9% ± 1.8% and 20.7% ± 2.5%, respectively) and approximately 15% enlargements of rS and rL. Thus, the change in LpS% represents a dramatic increase in the number of large pores and not an increased size of preexisting large pores, since the small and large pore radii changed in parallel. CONCLUSION(S): These results indicate that capillary permeability of the ovarian blood-follicle barrier is modulated by gonadotropin, mainly through increased numbers of large pores, similar to a classical inflammatory response.