Helen H. Kim

Insulin regulation of GnRH gene expression through MAP kinase signaling pathways

In mammals, reproduction is acutely regulated by metabolic status. Insulin is an important nutritional signal from the periphery that may regulate the reproductive axis. To determine whether insulin acts directly on the GnRH neuron, we performed studies in mouse-derived GnRH-expressing cell lines. Both insulin receptor protein and mRNA were detected in these cells. A saturation radioligand binding assay revealed high affinity, low capacity binding sites for insulin in GnRH neurons. Insulin also stimulated GnRH promoter activity in GnRH neurons. This effect was blocked by pretreatment with the MEK inhibitor, PD98059, indicating a role for MAP kinase signaling. In transient transfection studies, insulin treatment stimulated expression of a 1250 bp mouse GnRH gene promoter fragment four-fold when compared to promoter activity in untreated cells. In contrast, insulin did not stimulate activity of a 587 bp fragment of the mGnRH gene promoter, indicating that the promoter elements mediating insulin stimulation of the GnRH promoter are located between -1250 and -587 bp. Our studies suggest that insulin may regulate reproductive function by direct effects on the GnRH neurons and specifically by stimulating GnRH gene expression.

Promoter Sequences Targeting Tissue-specific Gene Expression of Hypothalamic and Ovarian Gonadotropin-releasing Hormone in Vivo

Molecular mechanisms directing tissue-specific expression of gonadotropin-releasing hormone (GnRH) are difficult to study due to the paucity and scattered distribution of GnRH neurons. To identify regions of the mouse GnRH (mGnRH) promoter that are critical for appropriate tissue-specific gene expression, we generated transgenic mice with fragments (−3446/+23 bp, −2078/+23 bp, and −1005/+28 bp) of mGnRH promoter fused to the luciferase reporter gene. The pattern of mGnRH promoter activity was assessed by measuring luciferase activity in tissue homogenates. All three 5′-fragments of mGnRH promoter targeted hypothalamic expression of the luciferase transgene, but with the exception of the ovary, luciferase expression was absent in non-neural tissues. High levels of ovarian luciferase activity were observed in mice generated with both −2078 and −1005 bp of promoter. Our study is the first to define a region of the GnRH gene promoter that directs expression to both neural and non-neural tissuesin vivo. We demonstrate that DNA sequences contained within the proximal −1005 bp of the mGnRH promoter are sufficient to direct mGnRH gene expression to both the ovary and hypothalamus. Our results also suggest that DNA sequences distal to −2078 bp mediate the repression of ovarian GnRH.

The Gonadotropin-Releasing Hormone Cell-Specific Element Is Required for Normal Puberty and Estrous Cyclicity

Appropriate tissue-specific gene expression of gonadotropin-releasing hormone (GnRH) is critical for pubertal development and maintenance of reproductive competence. In these studies, a common element in the mouse GnRH (mGnRH) promoter, between −2806 and −2078 bp, is shown to mediate differential regulation of hypothalamic and ovarian mGnRH expression. To further characterize this region, we generated a knock-out mouse (GREKO−/−) with a deletion of the mGnRH promoter fragment between −2806 and −2078 bp. GnRH mRNA expression in the brain of GREKO−/− was less than the expression in wild-type mice; however, immunohistochemical analysis revealed no difference between the numbers of GnRH neurons among groups. GnRH mRNA expression in the ovary was fivefold higher in GREKO−/−. The immunohistochemical staining for GnRH in the ovary increased in surface epithelial and granulosa cells and also in the corpora lutea of GREKO−/− mice. The reproductive phenotype revealed that the mean day of vaginal opening was delayed, and additionally, there was a significant decrease in the length of proestrus and diestrus-metestrus phases of the estrous cycle, resulting in a shortened estrous cycle in GREKO−/− mice. This work supports the hypothesis that the region of the GnRH promoter contained between −2806 and −2078 bp acts as a cell-specific enhancer in the GnRH neuron and as a repressor in the ovary. Deletion of this region in vivo implicates the GnRH promoter in mediating pubertal development and periodic reproductive cycling, and forms the foundation to define the nuclear proteins important for puberty and estrous cycling in mammals.

Treatment of infertility in women with pituitary tumors.

Due to the pituitary’s critical position, a pituitary tumor may disrupt gonadal function, either by its expanding size or the inappropriate secretion of hormones. Menstrual cycles may be disrupted even without frank hypogonadism, particularly in the case of hormone-secreting adenomas. Despite optimal medical and surgical management of pituitary tumors, ovulation-induction therapy with gonadotropins is often required to restore fertility in these women. This article will provide an overview of the therapeutic options available for women with infertility resulting from pituitary tumors. Treatment strategies including dopamine agonists, gonadotropins and the role of assisted reproductive technologies will be discussed. Unique pregnancy considerations in the female patient with hypopituitarism will also be addressed.

The GnRH neuron: molecular aspects of migration, gene expression and regulation.

Publisher Summary The lack of migration of gonadotropin-releasing hormone (GnRH) neurons into the forebrain results in reproductive dysfunction. The forms of idiopathic hypogonadotropic hypogonadism not associated with Kallmanns syndrome are described in humans, but the mechanisms of disregulation of the GnRH neurons remain unknown. A lack of developmental progression of GnRH neurons due to the absence of proteins important for migration results in some of the cases of idiopathic hypogonadotropic hypogonadism. A mouse model exists for a non-Kallmanns-like associated hypogonadotropic hypogonadism. It is unclear whether GnRH neurons are merely passively responding to signals in their immediate milieu or whether local signals trigger changes in gene expression in GnRH neurons that are ultimately responsible for migration and for changes in cellular morphology or GnRH gene expression. There is evidence for the latter model in another neuronal system: the developmental organization of the cerebellum. As a means of developing an in vitro model for the study of GnRH neuronal cell activity, immortalized GnRH-expressing neuronal cell lines are created by targeted tumorigenesis. The chapter also explains cell-specific expression of the GnRH gene.

Family Building by Same-Sex Male Couples via Gestational Surrogacy

&NA; Best estimates suggest that the number of households with same‐sex male couples is increasing. One option for family building by same‐sex male couples is gestational surrogacy. Embryos would be generated in vitro, using the biologic fathers sperm with donor oocytes, and another woman (the gestational carrier) would undergo an embryo transfer to bear a child. Conceiving via gestational surrogacy requires advance planning, not only to coordinate the oocyte donor and gestational carrier but also to comply with regulations set forth by the Food and Drug Administration (FDA). The American Society for Reproductive Medicine (ASRM) has also published recommendations for practices using gestational carriers, which, in many cases, are more stringent than the FDA regulations. This article will review the FDA regulations and ASRM recommendations and their implications for same‐sex male couples who plan to conceive via gestational surrogacy.

Surrogate Pregnancy After Prenatal Diagnosis of Spina Bifida

Some pregnancies today involve infertile individuals or couples who contract with a fertile woman to carry a pregnancy for them. The woman who carries the pregnancy is referred to as a “gestational carrier.” The use of such arrangements is increasing. Most of the time, these arrangements play out as planned; sometimes, however, problems arise. This article discusses a case in which a fetal diagnosis of spina bifida led the infertile couple to request that the gestational carrier terminate the pregnancy, and the gestational carrier did not wish to do so. Experts in the medical and legal issues surrounding surrogacy discuss the considerations that should go into resolving such a conflict.

Transcriptional Development of the Hypothalamic-Pituitary-Gonadal Axis

Unlike other hypothalamic neuropeptide-producing cells, GnRH neurons do not arise from the developing basal forebrain. They originate in the olfactory placode in mammals and migrate across the nasal cavity and cribiform plate into the forebrain (1, 2). In mice, for example, the GnRH neurons are derived from the neural ectoderm, are born on d 10.5 postcoitus (pc), and begin to express GnRH mRNA and protein between d 10.75 and 11.5 pc. GnRH neurons then migrate across the olfactory cavity to the forebrain between d 12.5 pc and d 16.5 pc (2, 3). GnRH neurons migrating across the nasal cavity appear to be guided, in part, by the polysialic acid-rich form of the neural cell adhesion molecule (PSA-NCAM) (4) in association with the vomeronasal nerves (VNN). Recent evidence in studies of chick forebrain development has further implicated PSA-NCAM as an important component in GnRH neuronal migration (5). Other investigators have posited that factors such as gamma aminobutyric acid (GABA) (6, 7), adhesion-related kinase (8), or peripherin (9) might play a role in the guidance of the GnRH neurons. The initial migration to the forebrain follows the vomeronasal and terminal nerves. However, after entering the rostral forebrain, GnRH neurons become disassociated from the caudal branch of the VNN. Therefore, it is unclear what guides them during the remaining portion of their migration to the basal hypothalamus. At this time, it is felt that GnRH neurons are no longer guided by a defined anatomical structure (10). Instead, neuronal migration may trail behind as axonal migration to the median eminence and organum vasculosum of the lamina terminalis (OVLT) is occurring.

Semen preparation with the Sperm Select system versus a washing technique*†‡*Sperm Select; Select Medical Systems, Williston, Vermont.†Supported by a grant from Select Medical Systems, Williston, Vermont.‡Presented at the 41st Annual Meeting of the Pacific Coast Fertility Society, Indian Wells, California, April 14 to 18, 1993.

OBJECTIVE To compare sperm migration through sodium hyaluronate with simple washing as methods for preparing sperm for IUI. DESIGN Ten normal semen specimens were prospectively collected and samples were prepared by simple washing and by migration into sodium hyaluronate using the Sperm Select System (Select Medical Systems, Williston, VT). The semen and each treatment group were evaluated for sperm concentration, percent motile, viability, acrosomal status, longevity, and computer-aided semen analysis (CASA) parameters. SETTING University reproductive endocrinology facility. RESULTS The recovery of motile sperm was significantly higher for the washing method (mean +/- SEM 75% +/- 7%) than for the hyaluronate method (10% +/- 1%). The number of motile sperm recovered by migration into hyaluronate was independent of the percentage of motile sperm in the semen specimen and positively correlated with sperm concentration. The hyaluronate method produced greater percentages of motile, viable, and morphologically normal sperm, with lower proportions of premature acrosome reactions, higher sperm velocity, and greater linearity. CONCLUSIONS The Sperm Select System method of sperm separation provides a highly uniform specimen with improved sperm quality. However, the recovery of motile sperm is considerably lower than for simple washing methods.