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.
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    The biology of mammalian spermatozoa in the oviduct
    ( 1990) Smith, Todd Timothy
    The oviduct occupies a unique position in mammalian reproduction as the site of sperm transport, the final maturation of sperm and egg, fertilization, and the initial development of the embryo. This dissertation examines the factors that control the number, distribution and physiological state of spermatozoa in the oviduct. The golden hamster (Mesocricetus auratus) was used as the animal model. The following summarizes my findings. The uterotubal junction restricts the passage of homologous and heterologous spermatozoa into the oviduct, furthermore, sperm motility is essential for efficient passage. After mating, spermatozoa rapidly enter the oviductal isthmus where they are stored. When mating occurs shortly after the onset of estrus, spermatozoa are stored for at least 8 h until near the time of ovulation. When mating occurs during ovulation, spermatozoa are stored for a minimum of 3 h. Spermatozoa stored in the isthmus during the preovulatory period do not become fully capacitated until near the time of ovulation. When mating occurs during ovulation, spermatozoa require a minimum of 3 h in the isthmus to become fully capacitated. Although many thousands of spermatozoa are stored in the isthmus, only a relatively small percentage of these spermatozoa survive. The spermatozoa that do survive attach to the oviductal mucosa during storage. Later, due to physiological changes in the sperm head plasma membrane that accompany capacitation, a small number of these spermatozoa detach from the mucosa and ascend to the ampulla to fertilize the eggs.
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    Morphology and physiology of Mammalian (hamster) eggs before and after fertilization
    ( 1989) Yang, Cheng-Hsiung
    Preovulatory mature ovarian hamster eggs were compared with recently ovulated eggs. Although these two groups of eggs were at metaphase of the second meiotic division, there were quantitative differences between the two. The most striking difference was found in the zona pellucida. The zona of the oviductal egg was "heterogeneous" in its optical density and had a stronger acrosome reaction-inducing ability than that of the ovarian egg. When cultured in artificial media, the ooplasm of ovarian eggs became like that of oviductal eggs. However, their zonae remained unchanged. Zonae of ovarian eggs became like those of oviductal eggs only when they were exposed to oviductal fluid. To investigate egg morphology after centrifugation, and how egg fragments respond to spermatozoa, zona-free hamster eggs were centrifuged in a Percoll gradient. Each egg was separated into a light and a heavy half. Chromosomes remained in their original cortical position in the egg during centrifugation or after separation into either light or heavy half. When the eggs were treated with cytochalasin D (CD) and then centrifuged, the chromosomes were extruded quickly before each egg was separated into halves or fragment. Fertilizability was different between light and heavy halves. Most of the heavy halves supported development of sperm nuclei into pronuclei, whereas only a few light halves could do so. When the light and heavy halves were centrifuged further, each separated into two quarters. The lightest quarter was fragile and few of them could fuse with spermatozoa. In this quarter, the sperm nucleus could decondense but could not develop into a pronucleus, whereas in the other quarters the sperm nuclei could develop into well-formed pronuclei. The rigidity of eggs at various stages were examined by determining the minimum time needed for complete separation of all eggs in a group into halves. The rigidity of the egg diminished sharply with the progression of meiotic maturation, reaching a minimum at metaphase' of second meiosis, then increasing progressively after fertilization. Cytochalasin D reduced the rigidity of any stages of eggs, indicating that the actin-based cytoskeleton in the cortical region is largely responsible for the rigidity of the egg.