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Hitoshi Ozawa, Fang Han, and Mitsuhiro Kawata

Growth hormone (GH) cells in the rat anterior pituitary have been morphologically classified into three subtypes: type I (mature) containing large secretory granules about 350 nm in diameter, type II (intermediate) containing a mixture of large and small granules, and type III (immature) containing small granules about 150 nm in diameter. However, the functional implications of morphological heterogeneity, especially the different sensitivities to growth hormone-releasing hormone (GRH) under different corticosteroid conditions have not been elucidated to date.

In the present study, by application of microwave irradiation (MWI) for fixation and immunocytochemistry, new findings of the exocytotic response have been revealed among the subsets of GH cells following adrenalectomy (ADX), corticosterone treatment and/or GRH treatment.

The MWI gave effective results for fixation, especially for the permeability of the fixative, and showed good results for immunoelectron microscopy using the protein-A gold method. Moreover, the use of MWI greatly shortened the fixation, processing and immunolabeling times without compromising the quality of ultrastructural preservation and the specificity of labeling.

The number of exocytotic figures was low in all subtypes of GH cells in the sham-operated control rats. GRH treatment induced a significant increase in exocytosis in each subtype of GH cells, particularly in type I (mature) and type II (intermediate) GH cells in the control rats. GRH injection to rats for 4 days after ADX also showed an increase in exocytosis, but the degree was significantly less in comparison with the GRH injection in the control group. Corticosterone replacement given to ADX rats induced a clear recovery of the exocytotic response to GRH to the control level. Serum GH content measured by radioimmunoassay correlated with these morphological results.

These results suggest that the secretion of GH stimulated by GRH is closely related to corticosteroids, and that the sensitivity to GRH differs among the three subtypes of GH cells.

Free access

Kinuyo Iwata, Yuyu Kunimura, Keisuke Matsumoto, and Hitoshi Ozawa

Hyperandrogenic women have various grades of ovulatory dysfunction, which lead to infertility. The purpose of this study was to determine whether chronic exposure to androgen affects the expression of kisspeptin (ovulation and follicle development regulator) or release of luteinizing hormone (LH) in female rats. Weaned females were subcutaneously implanted with 90-day continuous-release pellets of 5α-dihydrotestosterone (DHT) and studied after 10 weeks of age. Number of Kiss1-expressing cells in both the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC) was significantly decreased in ovary-intact DHT rats. Further, an estradiol-induced LH surge was not detected in DHT rats, even though significant differences were not observed between DHT and non-DHT rats with regard to number of AVPV Kiss1-expressing cells or gonadotrophin-releasing hormone (GnRH)-immunoreactive (ir) cells in the presence of high estradiol. Kiss1-expressing and neurokinin B-ir cells were significantly decreased in the ARC of ovariectomized (OVX) DHT rats compared with OVX non-DHT rats; pulsatile LH secretion was also suppressed in these animals. Central injection of kisspeptin-10 or intravenous injection of a GnRH agonist did not affect the LH release in DHT rats. Notably, ARC Kiss1-expressing cells expressed androgen receptors (ARs) in female rats, whereas only a few Kiss1-expressing cells expressed ARs in the AVPV. Collectively, our results suggest excessive androgen suppresses LH surge and pulsatile LH secretion by inhibiting kisspeptin expression in the ARC and disruption at the pituitary level, whereas AVPV kisspeptin neurons appear to be directly unaffected by androgen. Hence, hyperandrogenemia may adversely affect ARC kisspeptin neurons, resulting in anovulation and menstrual irregularities.

Free access

Shimpei Higo, Satoko Aikawa, Norio Iijima, and Hitoshi Ozawa

In mammals, lactation suppresses GnRH/LH secretion resulting in transient infertility. In rats, GnRH/LH secretion is rescued within 18–48 h after pup separation (PS) and rapidly re-suppressed by subsequent re-exposure of pups. To elucidate the mechanisms underlying these rapid modulations, changes in the expression of kisspeptin, a stimulator of GnRH secretion, in several lactating conditions (normal-lactating; 4-h PS; 18-h PS; 4-h PS +1-h re-exposure of pups; non-lactating) were examined using in situ hybridization. PS for 4 h or 18 h increased Kiss1 expressing neurons in both the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (ARC), and subsequent exposure of pups re-suppressed Kiss1 in the AVPV. A change in Kiss1 expression was observed prior to the reported time of the change in GnRH/LH, indicating that the change in GnRH/LH results from changes in kisspeptin. We further examined the mechanisms underlying the rapid modulation of Kiss 1. We first investigated the possible involvement of ascending sensory input during the suckling stimulus. Injection of the anterograde tracer to the subparafascicular parvocellular nucleus (SPFpc) in the midbrain, which relays the suckling stimulus, revealed direct neuronal connections between the SPFpc and kisspeptin neurons in both the AVPV and ARC. We also examined the possible involvement of prolactin (PRL). Administration of PRL for 1 h suppressed Kiss1 expression in the AVPV but not in the ARC. These results indicate that suckling stimulus rapidly modulates Kiss1 expression directly via neuronal connections and indirectly through serum PRL, resulting in modulation in GnRH/LH secretion.

Open access

Hiroyuki Enomoto, Kinuyo Iwata, Keisuke Matsumoto, Mai Otsuka, Akio Morita, and Hitoshi Ozawa

Kisspeptin neurons, i.e. KNDy neurons, in the arcuate nucleus (ARC) coexpress neurokinin B and dynorphin and regulate gonadotropin-releasing hormone/luteinizing hormone (LH) pulses. Because it remains unclear whether these neurons are associated with reproductive dysfunction in diabetic females, we examined the expression of KNDy neurons detected by histochemistry in streptozotocin (STZ)-induced diabetic female rats 8 weeks after STZ injection. We also evaluated relevant metabolic parameters – glucose, 3-hydroxybutyrate, and non-esterified fatty acids – as indicators of diabetes progression. Severe diabetes with hyperglycemia and severe ketosis suppressed the mRNA expression of KNDy neurons, resulting in low plasma LH levels and persistent diestrus. In moderate diabetes with hyperglycemia and moderate ketosis, kisspeptin-immunoreactive cells and plasma LH levels were decreased, while the mRNA expression of KNDy neurons remained unchanged. Mild diabetes with hyperglycemia and slight ketosis did not affect KNDy neurons and plasma LH levels. The number of KNDy cells was strongly and negatively correlated with plasma 3-hydroxybutyrate levels. The vaginal smear analysis showed unclear proestrus in diabetic rats 3–5 days after STZ injection, and the mRNA expression of kisspeptin in the ARC was decreased 2 weeks after STZ injection in severely diabetic rats. Kisspeptin neurons in the anteroventral periventricular nucleus (AVPV), which induce an LH surge, were unaffected at 2 and 8 weeks after STZ injection regardless of the diabetes severity. These results suggest that diabetes mellitus progression in females may negatively affect ARC kisspeptin neurons but not AVPV kisspeptin neurons, implicating a potential role of ARC kisspeptin neurons in menstrual disorder and infertility.