Luteinizing Hormone Action in Human Oocyte Maturation and Quality: Signaling Pathways, Regulation, and Clinical Impact.

The ovarian follicle luteinizing hormone (LH) signaling molecules that regulate oocyte meiotic maturation have recently been identified. The LH signal reduces preovulatory follicle cyclic nucleotide levels which releases oocytes from the first meiotic arrest. In the ovarian follicle, the LH signal reduces cyclic nucleotide levels via the CNP/NPR2 system, the EGF/EGF receptor network, and follicle/oocyte gap junctions. In the oocyte, reduced cyclic nucleotide levels activate the maturation promoting factor (MPF). The activated MPF induces chromosome segregation and completion of the first and second meiotic divisions. The purpose of this paper is to present an overview of the current understanding of human LH signaling regulation of oocyte meiotic maturation by identifying and integrating the human studies on this topic. We found 89 human studies in the literature that identified 24 LH follicle/oocyte signaling proteins. These studies show that human oocyte meiotic maturation is regulated by the same proteins that regulate animal oocyte meiotic maturation. We also found that these LH signaling pathway molecules regulate human oocyte quality and subsequent embryo quality. Remarkably, in vitro maturation (IVM) prematuration culture (PMC) protocols that manipulate the LH signaling pathway improve human oocyte quality of cultured human oocytes. This knowledge has improved clinical human IVM efficiency which may become a routine alternative ART for some infertile patients.

Expression of kv4.3 voltage-gated potassium channels in rat gonadotrophin-releasing hormone (GnRH) neurons during the estrous cycle.

Regular and timely electrical activity of gonadotrophin-releasing hormone (GnRH) neurons accompanies the pulsatile release of GnRH that plays a central role in regulating fertility. Although transient outward A-type currents (I(A)) have been electrophysiologically identified in GnRH neurons, the molecular identity of the channels that underlie these currents are unknown. Several families of voltage-gated potassium channels can underlie I(A). However, the biophysical properties of I(A) described in previous electrophysiological studies are strongly characteristic of members of the Kv4 family of voltage-gated channels. We, therefore, sought to determine the presence of Kv4 channels in GnRH neurons. We used reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis to determine whether Kv4 messenger RNA (mRNA) and protein are present in the rat medial preoptic area (MPOA) and median eminence (ME). We used double-label immunohistochemistry to determine whether Kv4 colocalized with GnRH cell bodies in the MPOA and GnRH axons in the ME. Kv4.3 channels co-localized with GnRH in the MPOA but not in the ME. Neither Kv4.2 nor Kv4.1 co-localized with GnRH in either the MPOA or the ME. The electrical activity of GnRH neurons changes dramatically during the estrous cycle. We, therefore, studied the change in Kv4.3 expression in GnRH neurons during the estrous cycle. In the estrus phase, 58.05% of GnRH neurons expressed Kv4.3 compared to 74.48% in diestrus-proestrus rats (P < .05). Our data suggest that Kv4.3 is the major molecular component of I(A) in GnRH neurons, and furthermore that the expression of Kv4.3 changes significantly during the rat estrous cycle.

The expression of hyperpolarization activated cyclic nucleotide gated (HCN) channels in the rat ovary are dependent on the type of cell and the reproductive age of the animal: a laboratory investigation.

BACKGROUND: Aim of this study was to test the hypothesis that levels of hyperpolarization activated cyclic nucleotide gated channels 1 to 4 (HCN1-4) are linked to the reproductive age of the ovary. METHODS: Young, adult, and reproductively aged ovaries were collected from Sprague-Dawley rats. RT-PCR and western blot analysis of ovaries was performed to investigate the presence of mRNA and total protein for HCN1-4. Immunohistochemistry with semiquantitative H score analysis was performed using whole ovarian histologic sections. RESULTS: RT-PCR analysis showed the presence of mRNA for HCN1-4. Western blot analysis revealed HCN1-3 proteins in all ages of ovarian tissues. Immunohistochemistry with H score analysis demonstrated distinct age-related changes in patterns of HCN1-3 in the oocytes, granulosa cells, theca cells, and corpora lutea. HCN4 was present only in the oocytes, with declining levels during the reproduction lifespan. CONCLUSION: The evidence presented here demonstrates cell-type and developmental age patterns of HCN1-4 channel expression in rat ovaries. Based on this, we hypothesize that HCN channels have functional significance in rat ovaries and may have changing roles in reproductive aging.

Serum and ovarian Müllerian inhibiting substance, and their decline in reproductive aging.

The objectives of this study were to identify whether there is a decline in Müllerian inhibiting substance (MIS) in the female rat during chronological aging, and to define the physiological basis of aging-related changes in MIS. The results demonstrate that there is an exponential decline in both serum and ovarian levels of MIS with increasing female age, and that the histologic origin for the reduction in serum levels of MIS is a decline in the number of small ovarian follicles expressing MIS.

Hyperpolarization-activated cation channels are expressed in rat hypothalamic gonadotropin-releasing hormone (GnRH) neurons and immortalized GnRH neurons.

OBJECTIVES: The current research was conducted to determine whether hyperpolarization-activated cyclic nucleotide-gated (HCN1-4) channels are expressed in gonadotropin-releasing hormone (GnRH) neurons in the female rat hypothalamus and immortalized GnRH neurons (GT1-7 cells). METHODS: Double-label fluorescence immunohistochemistry was used to colocalize HCN1-4 channels and GnRH in GnRH neurons in the female rat hypothalamus. Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemistry were used to analyze HCN channel gene expression in GT1-7 cells. RESULTS: Double-label fluorescence immunohistochemistry showed that 43% of hypothalamic GnRH neurons immunostained for HCN2 and 90% of GnRH neurons immunostained for HCN3. RT-PCR and Western blot showed expression of all four HCN channel subunits in GT1-7 cells. Double-label immunocytochemistry showed cytoplasmic immunostaining of HCN2 and HCN3 in GT1-7 cells. CONCLUSIONS: This study demonstrates for the first time that HCN channels are expressed in GnRH neurons in the rat hypothalamus and GT1-7 cells. Our research supports the hypothesis that HCN channels may be involved in electrical bursting activity and pulsatile GnRH secretion in endogenous GnRH neurons and GT1-7 cells.

A unique case of combined pituitary hormone deficiency caused by a PROP1 gene mutation (R120C) associated with normal height and absent puberty.

We report a 28-year-old-female who presented with primary amenorrhoea, absence of puberty, obesity and normal stature. The subject was clearly short as a child, with a height more than 2 SD below normal until the age of 15 years. The pubertal growth spurt failed to develop. She continued growing at a prepubertal rate until growth ceased at the age of 20 years, reaching her final adult height of 157 cm (SDS -0.86) without hormonal treatment. A combined pituitary hormone stimulation test of anterior pituitary function showed deficiencies of GH, LH and FSH, and low normal serum levels of TSH and PRL. Magnetic resonance imaging revealed a hypoplastic pituitary with markedly reduced pituitary height. In addition, a whole body dual energy X-ray absorptiometry scan showed high levels of body fat (54%). Combined pituitary hormone deficiencies with a hypoplastic pituitary suggested the diagnosis of a Prophet of Pit-1 (PROP1) gene mutation. Normal stature in this case, however, confounded this diagnosis. Sequencing of PROP1 revealed homozygosity for a single base-pair substitution (C to T), resulting in the replacement of an Arg by a Cys at codon 120 (R120C) in the third helix of the homeodomain of the Prop-1 protein. To our knowledge, this is the first report of a patient with a mutation in the PROP1 gene that attained normal height without hormonal treatment, indicating a new variability in the PROP1 phenotype, with important implications for the diagnosis of these patients. We suggest that this can be explained by (i) the presence of low levels of GH in the circulation during childhood and adolescence; (ii) the lack of circulating oestrogen delaying epiphyseal fusion, resulting in growth beyond the period of normal growth; and (iii) fusion of the epiphyseal plates, possibly as a result of circulating oestrogens originating from peripheral conversion of androgens by adipose tissue.

A role for hypothalamic astrocytes in dehydroepiandrosterone and estradiol regulation of gonadotropin-releasing hormone (GnRH) release by GnRH neurons.

Molecules of astrocyte origin influence gonadotropin-releasing hormone (GnRH) release and GnRH neuronal growth and differentiation. Furthermore, type 1 astrocytes express steroid receptors, presenting the possibility that steroid actions on GnRH neurons might occur via astrocytes. Utilizing GT1-7 cells, a GnRH-secreting cell line, the present study demonstrates that astrocytes mediate dehydroepiandrosterone (DHEA) or estradiol (E2) stimulated GnRH secretion. Conditioned media (CM) from astrocytes cultured for 48 h alone, with DHEA (DHEA-CM), or with E2 (E2-CM) were collected, treated with charcoal to remove steroids, and added to GT1-7 cells in culture for 12 h to test the effect on GnRH secretion. DHEA-CM and E2-CM stimulated GnRH secretion by GT1-7 cells by 4- and 3-fold, respectively. The effect of DHEA-CM on GnRH secretion by GT1-7 cells appears to be related to both DHEA and its metabolite, E2, since blocking the metabolism of DHEA into estrogen in the DHEA-treated astrocytes partially reversed the stimulatory effect of DHEA-CM. Addition of transforming growth factor (TGF)-beta1-neutralizing antibody to the astrocyte cultures reversed the stimulatory effects of both DHEA-CM and E2-CM on GnRH secretion by GT1-7 cells, suggesting that TGF-beta1 derived from astrocytes may be the principle mediator of E2 and DHEA effects. These data provide evidence for a novel mechanism by which circulating steroids and/or neurosteroids may modulate GnRH secretion.

Utilidad de la ecocardiografía transesofágica en el diagnóstico de tumores intracardiacos y extracardiacos / The use of transesophageal echocardiography in the diagnosis of intracardiac and extracardiac tumors

De enero de 1990 a agosto de 1993, se estudiaron 18 pacientes con sospecha de masa intratorácica. En todos se efectuó ECG, radiografía de tórax y ecocardiograma transtorácico y transesofágico. Se dividieron en dos grupos. Grupo I: ocho pacientes con mixoma intracardiaco. Grupo II: diez pacientes con tumor no mixoma. La naturaleza histopatológica del tumor intra o extra cardiaco se corroboró en todos los pacientes del Grupo I y en 8 casos del Grupo II. En el Grupo I, la edad promedio fue de 39 años y predominó el sexo femenino (7.5 por ciento). Con registros transtorácicos y transesofágicos, se demostró que en cuatro pacientes el mixoma se ubicaba en el atrio derecho, en otros en el atrio izquierdo, y en el restante ocupaba las cuatro cavidades cardiacas. En el Grupo II la edad promedio fue de 36 años y predominó el sexo masculino (60 por ciento). Con ecocardiografía se detectaron tumores cardiacos en 3 pacientes: se localizaron en las cavidades derechas (leiomioma y leiomiosarcoma); en el restante no fue posible reconocer la estirpe histológica del tumor auricular izquierdo. En los otros 7 pacientes con tumor mediastinal, se logró precisar la falta de invasión tumoral del corazón. Por los resultados obtenidos podemos concluir que la ecocardiografía transtorácica permite diagnosticar los tumores intracardiacos. En estos pacientes, los estudios transesofágicos brindan información adicional acerca del estado valvular, del sitio de implantación tumoral y de su infiltración parietal. En los tumores mediastinales, dichos estudios ofrecen mayor información sobre la potencial compresión cardiaca