Ph.D. - Biomedical Sciences (Anatomy and Reproductive Biology)

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    Gonadal steroid hormone regulation of hypothalamic opioid function
    ( 1994) Cheung, Sun
    β-Endorphin- (β-Endo) like immunoreactive (IR) fibers in the medial preoptic area (MPOA) have been shown to vary in density across the estrous cycle. The proopiomelanocortin (POMC) neurons which produce this innervation of the MPOA are located in the arcuate nucleus (ARC). The POMC mRNA level in the ARC neurons also varies across the estrous cycle. However, the effects of gonadal steroid hormones on the distribution and density of β-endo-like IR fibers in the MPOA, and on the regulation of POMC gene expression in ARC neurons which project to the MPOA of ovariectomized (OVX) female rats are unclear. In the present studies, immunohistochemical staining, and combined fluorogold (FG) retrograde labeling and in situ hybridization histochemistry were used to investigate these unknown problems. In the MPOA, the density of β-endo-like IR fibers was low in OVX animals, increased slightly following 17β-estradiol (E2) treatment or 3 hr after progesterone (P) injection. However, β-endo-like IR fiber density increased remarkably 27 hr after E2P treatment, and remained elevated 51 hr after E2P treatment POMC mRNA expression was low in ARC neurons of OVX animals, significantly increased in the rostral ARC 48 hr after E2 treatment, and P administration enhanced the E2 effect in rostral ARC neurons. In the other ARC regions, E2P significantly increased POMC mRNA beginning 13 hr after P injection. E2P treatment did not produce more POMC-containing fibers to innervate the MPOA. These results suggest that the density of MPOA β-endo-like IR fibers is gonadal steroid hormone-dependent, but the pattern of innervation of the MPOA by POMC neurons is unaffected by gonadal steroid hormone treatment. Gonadal steroid hormones appear to activate POMC expression in ARC neurons, stimulate the synthesis of β-endo in POMC neurons which project to the MPOA, and eventually promote transport of β-endo from POMC neurons in the ARC to the β-endo-containing axons in the MPOA. That steroid hormones functionally affect ARCPOMC mRNA expression and MPOA B-endo density implicates the roles of this endogenous opioid in regulating hormonal cyclicity and reproductive behavior.
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    Purine involvement in corpus luteum function in non-pregnant sheep
    ( 1992) Patrick, Kimberly Miller
    Five experiments were done to discern the involvement of de novo synthesis of purines on ovine corpus luteum (CL) function, and to establish a model for assessing the in vivo effects of purines on CL function. In order to identify specific purine involvements, the adenylic, guanylic, and inosinic biosynthesis pathways were isolated using the de novo synthesis pathway inhibitors hadacidin, mycophenolic acid, and azaserine. In three in vivo experiments, these treatments were delivered into the sheath surrounding the ovarian vascular pedicle (OVP) ipsilateral to the ovary bearing the corpus hemorrhagicum via an exteriorized indwelling catheter. Delivery of the drugs to the CL by uptake into the vasculature was confirmed indirectly by HPLC analysis of luteal AMP, IMP, and GMP levels. Azaserine, hadacidin, or mycophenolic acid, or these drugs plus replacement compounds (inosine, adenosine, or guanosine, respectively), were delivered in phosphate buffered saline (PBS) via the OVP catheter at 4 or 6 hour intervals over days 1-7 or 1-8 post-estrus. Daily jugular blood samples were taken over the treatment period and quantified for estradiol-17γ and progesterone by RIA. CL, collected on day 7 or 8 post-estrus, were dissociated, and luteal cell populations and live/dead ratios of luteal cells were noted. Augmented profiles of progesterone and estradiol-17 γ were seen in ewes treated with 150 µg of azaserine, or mycophenolic acid, respectively, as compared to ewes treated with 500 µg of any of the drugs or controls. Estradiol-17γ and progesterone profiles for drug + replacement compound-treated animals were not different from control ewes. In vitro experiments involving incubation of luteal slices in PBS + luteinizing hormone (LH) with azaserine alone or combined with one of the pathway replacement compounds revealed that azaserine-treated luteal slices were able to produce progesterone levels that were not different from those produced either by controls, or adenosine-treated slices (amplifies LH-stimulated progesterone production). These data suggest that either a) the CL is less dependent on de novo synthesized purines than the readily available salvage pathway-derived purines, or that b) there may be a non-purinergic dependent second messenger system controlling biosynthesis of steroids in luteal cells.
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    A study of the guinea pig relaxin gene(s)
    ( 1991) Yee, Lee
    This dissertation study was designed to elucidate the nucleotide sequence of the guinea pig relaxin gene and hence to derive the amino acid sequence of guinea pig preprorelaxin, to show how many relaxin gene(s) are present in the guinea pig genome, and to study the transcription of a relaxin gene in the guinea pig endometrium during the reproductive cycle. A lactating guinea pig mammary gland cDNA library was screened with a radioactive full-length rat preprorelaxin cDNA probe. Seven positive clones were identified. The characterization of the inserts with the PCR (Polymerase Chain Reaction) technique and Southern hybridization suggested that they are truncated molecules. In order to obtain general information of the guinea pig relaxin gene, the clone containing the longest insert (approximately 600 bps) was submitted to sequence analysis. Computer-assisted sequencing data analysis show that this insert was similar to the mRNA sequence of the pig preprorelaxin. The conclusion from this study was that the guinea pig relaxin gene sequence is similar to that of the pig relaxin gene. Based on the preliminary information from these studies, several sets of oligonucleotide primers were selected from different regions of the mRNA sequence of pig preprorelaxin. These synthetic primers were used to screen a cDNA "pool" prepared from the guinea pig endometrium in late pregnancy by a PCR technique. One set of these PCR primers successfully amplified a relaxin related cDNA fragment (286 bps). Sequence analysis of this PCR product confirmed that it encodes an intact B chain of the guinea pig preprorelaxin plus part of the signal peptide and C peptide of this molecule. The rest part of the guinea pig endometrial relaxin gene was elucidated by a RACE-PCR and subcloning strategy. This is the first report of any part of the guinea pig relaxin gene sequence. Availability of this sequence allowed a study of the physiology of guinea pig relaxin. The second question of this dissertation was studied by a Southern analysis of guinea pig genomic DNA digests with a guinea pig relaxin specific cDNA probe. It was shown that there are two different relaxin genes in the guinea pig genome, a situation similar to the human genome, but different to the genomes of other mammals studied thus far. One relaxin gene has been shown to be expressed in the guinea pig endometrium in this dissertation using PCR and direct sequencing. Whether ,or where, the second relaxin gene is expressed needs further investigation. The availability of the guinea pig relaxin gene sequence allowed a more convincing study of the expression of this gene in the guinea pig endometrium. Guinea pig endometrial mRNAs from different stages of the reproductive cycle were hybridized with the radioactive guinea pig relaxin cDNA probe. Northern analyses obtained from this study showed that the transcription pattern of the relaxin gene in this organ is maximal in the late pregnancy, minimal during mid pregnancy, appears in the estrous cycle and disappears rapidly after parturition. This is the first study of the transcription of the relaxin gene in the guinea pig endometrium during the reproductive cycle with a species-specific cDNA probe. It provides proof that relaxin is indeed synthesized in the guinea pig endometrium, not sequestered from other sources. The loss of transcription activity of the relaxin gene, in the endometrium, during lactation indicates that there are other sites of relaxin synthesis as sources of the plasma relaxin found in lactation.
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    Human relaxin, prolactin and placental lactogen in human intrauterine tissues
    ( 1991) Sakbun, Vannara
    The human placenta, fetal membranes and decidua can be classified as endocrine glands because of their abilities to produce hormones which maintain and ensure the success of a pregnancy. These hormones may enter the maternal and/or the fetal circulatory systems to act upon distant targets while others may be produced and act locally within the intrauterine compartment. This study was designed to look at three circulatory hormones, relaxin, prolactin and placental lactogen, as bipolar hormones, local and distant. The sources of their production were studied in five different intrauterine tissues at two physiological time frames, before and after labor. The corpus luteum is the source of circulating relaxin during pregnancy. To determine whether this hormone is produced locally in intrauterine tissues, two techniques have been used, immunocytochemistry and Northern analyses. An antiserum to a synthetic l4-amino acid sequence of the connecting peptide of human relaxin and two monoclonal antibodies to human relaxin were used to immunostain fetal membranes with adherent decidua and placental trophoblast. Poly(A)+RNA prepared from five separate tissues, the amnion, chorionic membrane, decidua parietalis , basal plate and the placental trophoblast were hybridized to a 48-mer oligoprobe to human relaxin. Results from both techniques showed that the decidua parietalis and basalis, the chorion and the placental trophoblast synthesize and produce relaxin. The mRNA species in the placental trophoblast was shown to be 1.1kb, about 100 base pairs smaller than the mRNA species in other tissues, suggesting different processing mechanisms for these two mRNA species for relaxin. Comparative quantitation of mRNA levels showed that the decidua parietalis expressed the gene for relaxin more than the other tissues. Also all tissues obtained after normal spontaneous delivery had a lower capability for relaxin synthesis than tissues obtained from term elective Cesarean section. It is generally accepted that the decidua parietalis is the primary source of amniotic fluid prolactin. Whether this hormone is a product of other intrauterine tissues has not been thoroughly defined. Two polyclonal and four monoclonal antibodies were used to localize human prolactin (hPRL) in human intrauterine tissues. Northern analyses using a 48-mer oligoprobe and a 712 base pair cDNA probe for hPRL were performed to distinguish synthesized from sequestered hormone. Results showed that the decidua parietalis is indeed the major source of amniotic fluid prolactin and that the chorion laeve and the basal plate are additional sources. There was no significant difference in hPRL mRNA levels between Cesarean section and normal vaginal delivery tissues. Human placental lactogen (hPL) is one of the major hormones secreted by the placental syncytiotrophoblast and is readily detected in the maternal circulation. In this study, a specific polyclonal antibody to hPL, a 48-mer oligoprobe and a 540 base pair cDNA probe to hPL were used to investigate and determine other possible intrauterine sources of this hormone. Results showed unequivocally that the syncytiotrophoblast is the classical source of hPL. In addition, some cells of the chorionic cytotrophoblast and the basal plate also synthesized hPL. Quantitative analysis on Northern blots showed that the mRNA levels for hPL in these extra-sources were less than one percent of that in the classical source syncytiotrophoblast. It is not known whether these small amounts of hPL by these ectopic sources stay and function locally in the intrauterine tissues or whether they contribute to maternal circulation. The differential production of the three hormones by intrauterine tissues presented in this dissertation provide further definition of a paracrine/autocrine system within these tissues.