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Sirolimus, also known as rapamycin, and its closely related rapamycin analog (rapalog) Everolimus inhibit "mammalian target of rapamycin complex 1" (mTORC1), whose activity is required for spermatogenesis. Everolimus is Food and Drug Administration approved for treating human patients to slow growth of aggressive cancers and preventing organ transplant rejection. Here, we test the hypothesis that rapalog inhibition of mTORC1 activity has a negative, but reversible, impact upon spermatogenesis. Juvenile (P20) or adult (P>60) mice received daily injections of sirolimus or Everolimus for 30 days, and tissues were examined at completion of treatment or following a recovery period. Rapalog treatments reduced body and testis weights, testis weight/body weight ratios, cauda epididymal sperm counts, and seminal vesicle weights in animals of both ages. Following rapalog treatment, numbers of differentiating spermatogonia were reduced, with concomitant increases in the ratio of undifferentiated spermatogonia to total number of remaining germ cells. To determine if even low doses of Everolimus can inhibit spermatogenesis, an additional group of adult mice received a dose of Everolimus ∼6-fold lower than a human clinical dose used to treat cancer. In these animals, only testis weights, testis weight/body weight ratios, and tubule diameters were reduced. Return to control values following a recovery period was variable for each of the measured parameters and was duration and dose dependent. Together, these data indicate rapalogs exerted a dose-dependent restriction on overall growth of juvenile and adult mice and negative impact upon spermatogenesis that were largely reversed; following treatment cessation, males from all treatment groups were able to sire offspring., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Aims: Intracardiac injection of recombinant EphrinA1-Fc immediately following coronary artery ligation in mice reduces infarct size in both reperfused and non-reperfused myocardium, but the cellular alterations behind this phenomenon remain unknown., Main Methods: Herein, 10 wk-old B6129SF2/J male mice were exposed to acute ischemia/reperfusion (30minI/24hrsR) injury immediately followed by intracardiac injection of either EphrinA1-Fc or IgG-Fc. After 24 h of reperfusion, sections of the infarct margin in the left ventricle were imaged via transmission electron microscopy, and mitochondrial function was assessed in both permeabilized fibers and isolated mitochondria, to examine mitochondrial structure, function, and energetics in the early stages of repair., Key Findings: At a structural level, EphrinA1-Fc administration prevented the I/R-induced loss of sarcomere alignment and mitochondrial organization along the Z disks, as well as disorganization of the cristae and loss of inter-mitochondrial junctions. With respect to bioenergetics, loss of respiratory function induced by I/R was prevented by EphrinA1-Fc. Preservation of cardiac bioenergetics was not due to changes in mitochondrial JH 2 O 2 emitting potential, membrane potential, ADP affinity, efficiency of ATP production, or activity of the main dehydrogenase enzymes, suggesting that EphrinA1-Fc indirectly maintains respiratory function via preservation of the mitochondrial network. Moreover, these protective effects were lost in isolated mitochondria, further emphasizing the importance of the intact cardiomyocyte ultrastructure in mitochondrial energetics., Significance: Collectively, these data suggest that intracardiac injection of EphrinA1-Fc protects cardiac function by preserving cardiomyocyte structure and mitochondrial bioenergetics, thus emerging as a potential therapeutic strategy in I/R injury., (Published by Elsevier Inc.)

Key Points: Breast cancer 1 early onset gene codes for the DNA repair enzyme, breast cancer type 1 susceptibility protein (BRCA1). The gene is prone to mutations that cause a loss of protein function. BRCA1/Brca1 has recently been found to regulate several cellular pathways beyond DNA repair and is expressed in skeletal muscle. Skeletal muscle specific knockout of Brca1 in mice caused a loss of muscle quality, identifiable by reductions in muscle force production and mitochondrial respiratory capacity. Loss of muscle quality was associated with a shift in muscle phenotype and an accumulation of mitochondrial DNA mutations. These results demonstrate that BRCA1 is necessary for skeletal muscle function and that increased mitochondrial DNA mutations may represent a potential underlying mechanism., Abstract: Recent evidence suggests that the breast cancer 1 early onset gene (BRCA1) influences numerous peripheral tissues, including skeletal muscle. The present study aimed to determine whether induced-loss of the breast cancer type 1 susceptibility protein (Brca1) alters skeletal muscle function. We induced genetic ablation of exon 11 in the Brca1 gene specifically in the skeletal muscle of adult mice to generate skeletal muscle-specific Brca1 homozygote knockout (Brca1KO smi ) mice. Brca1KO smi exhibited kyphosis and decreased maximal isometric force in limb muscles compared to age-matched wild-type mice. Brca1KO smi skeletal muscle shifted toward an oxidative muscle fibre type and, in parallel, increased myofibre size and reduced capillary numbers. Unexpectedly, myofibre bundle mitochondrial respiration was reduced, whereas contraction-induced lactate production was elevated in Brca1KO smi muscle. Brca1KO smi mice accumulated mitochondrial DNA mutations and exhibited an altered mitochondrial morphology characterized by distorted and enlarged mitochondria, and these were more susceptible to swelling. In summary, skeletal muscle-specific loss of Brca1 leads to a myopathy and mitochondriopathy characterized by reductions in skeletal muscle quality and a consequent kyphosis. Given the substantial impact of BRCA1 mutations on cancer development risk in humans, a parallel loss of BRCA1 function in patient skeletal muscle cells would potentially result in implications for human health., (© 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.)

In mammals, most neonatal male germ cells (prospermatogonia) are quiescent and located in the center of the testis cords. In response to an unknown signal, prospermatogonia transition into spermatogonia, reenter the cell cycle, divide, and move to the periphery of the testis cords. In mice, these events occur by 3-4 days postpartum (dpp), which temporally coincides with the onset of retinoic acid (RA) signaling in the neonatal testis. RA has a pivotal role in initiating germ cell entry into meiosis in both sexes, yet little is known about the mechanisms and about cellular changes downstream of RA signaling. We examined the role of RA in mediating the prospermatogonia-to-spermatogonia transition in vivo and found 24 h of precocious RA exposure-induced germ cell changes mimicking those that occur during the endogenous transition at 3-4 dpp. These changes included: 1) spermatogonia proliferation; 2) maturation of cellular organelles; and 3), expression of markers characteristic of differentiating spermatogonia. We found that germ cell exposure to RA did not lead to cellular loss from apoptosis but rather resulted in a delay of ∼2 days in their entry into meiosis. Taken together, our results indicate that exogenous RA induces multiple hallmarks of the transition of prospermatogonia to spermatogonia prior to their entry into meiosis.

Smooth muscle cells maintain filaments of actin and myosin in the presence of ATP, although dephosphorylated myosin filaments and actin-myosin interactions are unstable under those conditions in vitro. Several proteins that stabilize myosin filaments and that stabilize actin-myosin interactions have been identified. Fesselin or synaptopodin 2 appears to be another such protein. Rapid kinetic measurements and electron microscopy demonstrated that fesselin, isolated from turkey gizzard muscle, reduced the rate of dissociation of myosin filaments. Addition of fesselin increased both the length and thickness of myosin filaments. The rate of detachment of myosin, but not heavy meromyosin, from actin was also greatly reduced by fesselin. Data from this study suggest that fesselin stabilizes myosin filaments and tethers myosin to actin. These results support the view that one role of fesselin is to organize contractile units of myosin and actin.

In the testis, F-actin structures are involved in spermatid nuclear remodeling and cytoplasm reduction, maintenance of the blood-testis barrier, support of the spermatogonial stem cell niche, and release of spermatids into the tubular lumen. To gain a better understanding of actin regulation in Sertoli-germ cell interactions, we investigated the expression of the Palladin (Palld) gene, which encodes a widely expressed phosphoprotein that localizes to actin-rich cytoplasmic structures, including focal adhesions, cell-cell junctions, podosomes, and stress fibers, and serves as a molecular scaffold to bundle actin fibers. In germ cells, PALLD was concentrated along the tubulin- and F-actin-containing cytoplasmic manchette that forms adjacent to the elongating spermatid nucleus during spermiogenesis. To our surprise, PALLD relocated from the cytoplasm to the nucleus of Sertoli cells in the juvenile testis, coincident with the onset of puberty, and this localization was maintained in the adult. We provide evidence that the 140 kDa isoform of PALLD predominates in Sertoli cells, and that it is apparently cleaved, with the C-terminus localizing to the nucleus while the N-terminus remains cytoplasmic. We investigated the nuclear localization of the C-terminus of PALLD and found that it is regulated by a putative nuclear export signal. These results provide the foundation for future work employing Sertoli cell- and spermatid-specific Palld-knockout mice to study diverse roles of PALLD as both a nuclear-actin regulatory protein and as a potential regulator of manchette formation during spermatogenesis., (Copyright © 2013 Wiley Periodicals, Inc.)

This report compares cellular localization of fesselin in chicken smooth, skeletal and cardiac muscle tissues using affinity purified polyclonal fesselin antibodies. Western blot analyses revealed large amounts of fesselin in gizzard smooth muscle with lower amounts in skeletal and cardiac muscle. In gizzard, fesselin was detected by immunofluorescence as discrete cytoplasmic structures. Fesselin did not co-localize with talin, vinculin or caveolin indicating that fesselin is not associated with dense plaques or caveolar regions of the cell membrane. Immunoelectron microscopy established localization of fesselin within dense bodies. Since dense bodies function as anchorage points for actin and desmin in smooth muscle cells, fesselin may be involved in establishing cytoskeletal structure in this tissue. In skeletal muscle, fesselin was associated with desmin in regularly spaced bands distributed along the length of muscle fibers suggesting localization to the Z-line. Infrequently, this banding pattern was observed in heart tissue as well. Localization at the Z-line of skeletal and cardiac muscle suggests a role in contraction of these tissues.

Males of advanced age represent a rapidly growing population at risk for prostate cancer. In the contemporary setting of earlier detection, a majority of prostate carcinomas are still clinically localized and often treated using radiation therapy. Our recent studies have shown that premature cellular senescence, rather than apoptosis, accounts for most of the clonogenic death induced by clinically relevant doses of irradiation in prostate cancer cells. We show here that this treatment-induced senescence was associated with a significantly increased release of exosome-like microvesicles. In premature senescence, this novel secretory phenotype was dependent on the activation of p53. In addition, the release of exosome-like microvesicles also increased during proliferative senescence in normal human diploid fibroblasts. These data support the hypothesis that senescence, initiated either by telomere attrition (e.g., aging) or DNA damage (e.g., radiotherapy), may induce a p53-dependent increase in the biogenesis of exosome-like vesicles. Ultrastructural analysis and RNA interference-mediated knockdown of Tsg101 provided significant evidence that the additional exosomes released by prematurely senescent prostate cancer cells were principally derived from multivesicular endosomes. Moreover, these exosomes were enriched in B7-H3 protein, a recently identified diagnostic marker for prostate cancer, and an abundance of what has recently been termed "exosomal shuttle RNA." Our findings are consistent with the proposal that exosomes can transfer cargos, with both immunoregulatory potential and genetic information, between cells through a novel mechanism that may be recruited to increase exosome release during accelerated and replicative cellular senescence.

Tight junctions (TJ) constitute paracellular diffusion channels regulating the passage of ions and solutes across epithelia. We recently demonstrated that overexpression of the TJ membrane protein claudin-7 in LLC-PK1 cells decreases paracellular permeability to Cl(-) and increases paracellular permeability to Na(+). To investigate the effect of charged amino acid residues in extracellular domains (ED) of claudin-7 on paracellular charge selectivity, we created claudin-7 mutants by replacing negatively charged amino acids on ED with positively charged amino acids. Immunofluorescence light microscopy showed that these mutant proteins were correctly targeted to the cell junction. Ultrastructure examination of TJ morphology did not reveal any difference between cells expressing wildtype (WT) and mutant claudin-7. However, electrophysiological studies showed increased Cl(-) permeability in cells expressing first extracellular domain (ED1) mutants, but not second extracellular domain (ED2) mutants, compared to that of WT claudin-7. Our results demonstrate that negatively charged amino acids in ED1 of claudin-7 are involved in modulating paracellular Cl(-) permeability.

A homologous hamster relaxin RIA was developed to evaluate plasma and tissue concentrations of relaxin in the latter half of pregnancy in this species. Relaxin protein and mRNA were localized using antibodies developed to synthetic hamster relaxin and gene-specific molecular probes, respectively. Molecular weight and isoelectric point of the synthetic and native hormones were identical by electrophoretic methods, and synthetic hamster relaxin was active in the mouse interpubic ligament bioassay. Synthetic hormone was used as tracer and standard with rabbit antiserum to the synthetic hormone in the RIA. Relaxin was assayed in blood samples recovered from the retro-orbital plexus on Days 6, 8, 10, 12, 14, 15, and 16 of gestation and on Days 1 and 5 postpartum. Relaxin was first detected on Day 8 of gestation (3.7 +/- 0.6 ng/ml), increased to reach a maximum in the evening of Day 15 (826.0 +/- 124.0 ng/ml), and decreased by Day 16 (day of parturition). Relaxin concentrations were assayed in aqueous extracts of implantation sites (Days 6, 8, and 10) and chorioallantoic placentae (Days 12, 14, and 15). Concentrations were low on Day 6 (0.02 +/- 0.001 microg/g tissue), increased to Day 15 (6.96 +/- 0.86 microg/g tissue), and subsequently declined by the evening of Day 15. Relaxin protein and mRNA were localized to primary and secondary giant trophoblast cells in the chorioallantoic placental trophospongium. However, relaxin protein was not localized in ovaries of pregnant animals or oviductal tissues of cycling animals. Significant quantities of relaxin were detected in the serum of fetal hamsters recovered on Day 15.

Tissue distribution and cellular localization of PC1/3 mRNA in porcine tissues were examined by ribonuclease protection assay and in situ hybridization. PC1/3 mRNA was detected mainly in the corpus luteum of pregnant sow and brain. Within the ovary, PC1/3 and relaxin transcripts colocalized within large luteal cells. Levels of PC1/3 transcripts in corpora lutea increased as gestation advanced, parallel with an observed increase in relaxin transcripts. A role for PC1/3 in proprotein processing in the ovary is discussed.

In the hamster, serum total lactogenic activity increases during the latter half of gestation (Days 8-16). On Days 10 and 12 a substantial amount of lactogenic activity cannot be attributed to prolactin (PRL) and hamster placental lactogen-II (haPL-II); therefore, the presence of a molecule similar to placental lactogen-I (PL-I), as found in the rat and mouse at midpregnancy, has been hypothesized for the hamster. The objectives of this study were to identify PRL-like molecules synthesized by the hamster placenta and to determine the temporal and cellular synthesis of identified molecules. Oligonucleotides (20-23 bp) corresponding to regions of nucleotide homology between mouse PL-I (mPL-I) and rat PL-I (rPL-I) along with midgestation hamster placental RNA were used in 3' rapid amplification of cDNA ends (RACE) methodology to generate PRL-like cDNA. A 444-bp cDNA fragment that had nucleotide sequence similarity with members of the prolactin-growth hormone (PRL-GH) gene family was generated. This cDNA fragment was utilized to screen a Day 16 hamster placental bacteriophage cDNA library, and a clone containing the entire coding region was identified and sequenced. The molecule had 77% nucleotide sequence homology with mouse proliferin-related protein (mPRP) and somewhat less homology (approximately 60%) with hamster, rat, and mouse PRL or placental lactogens (PL). The derived amino acid sequence of the identified molecule contained a 15-residue signal sequence and a 219-residue peptide with a calculated molecular weight of 25477. The peptide shared 58% amino acid sequence identity with mPRP. Placental expression of the PRL-like molecule during the latter half of gestation was evaluated by Northern and slot-blot analyses using the 444-bp cDNA fragment as a hybridization probe. A 1-kb transcript was detected on Days 9-15 with peak expression on Days 10 and 11. Messenger RNA for the PRL-like molecule was localized to cytotrophoblast but not giant trophoblast cells of the placental trophospongium region. In addition, specific immunostaining using an antibody to mPRP was confined to cytotrophoblast cells.

The objectives of this study were to determine the nucleotide and derived amino acid sequences of hamster prorelaxin and to evaluate prorelaxin mRNA expression during gestation. Total RNA was extracted from tissues through use of guanidinium hydrochloride methodology. A 23-bp oligonucleotide pool derived from the N-terminal amino acid sequence of hamster prorelaxin and Day-14 placental RNA were used in 3' RACE methodology to generate a prorelaxin-specific cDNA. This cDNA fragment (940 bp) was utilized to screen a Day-16 hamster placental cDNA library, and a clone containing the entire coding region was identified. Nucleotide sequence analysis revealed a 531-bp open reading frame for preprorelaxin. The derived amino acid sequence contained a 22 amino acid signal peptide followed by a 155 amino acid prorelaxin sequence with a calculated molecular weight of 17,500. The derived prorelaxin amino acid sequence had 51.8%, 42.9%, 41.7%, and 38.2% homology with rat, human-1, human-2, and pig prorelaxins, respectively. Expression of prorelaxin mRNA during the latter half of gestation was evaluated by Northern and/or slot-blot analysis using the 940-bp cDNA fragment as a hybridization probe. Prorelaxin mRNA was first detected in the placenta on Day 8. Levels of the 900-bp transcript increased to a plateau on Days 10, 12, and 14 and subsequently increased further on Day 15. Prorelaxin mRNA was not detected in ovary, hippocampus, or neocortex.

The purpose of this investigation was to identify the cell types synthesizing hamster placental lactogen-II (haPL-II) in the chorioallantoic placenta. In addition, temporal changes in haPL-II mRNA expression, in placental mass, and in the proportion of haPL-II-containing cells per unit area were examined. Hamster PL-II mRNA expression was assessed by in situ hybridization and Northern and slot-blot analyses. Cells containing haPL-II protein were identified by immunocytochemical techniques. Placentae were recovered in the morning on Days 6, 8, 10, 12, and 14 of gestation and in the afternoon on Day 15. A single 1-kb haPL-II transcript was first detected on Day 10 of gestation. Hamster PL-II message increased to Day 14 and remained elevated on Day 15. Giant trophoblast cells (GTC) located in the trophospongium were the major source of haPL-II mRNA and protein, although GTC along the periphery of the placenta and in the labyrinth expressed haPL-II as well. Placental weight, trophospongium portion of placental weight, and number of haPL-II-immunostained GTC per unit of placental area increased between Days 12 and 14 and remained elevated to Day 15. These observations indicate that an increased number of haPL-II-producing GTC is a major factor contributing to increased maternal haPL-II serum levels during pregnancy.

Previous immunological studies have indicated that the molecular structure of hamster relaxin is quite different from that of porcine relaxin. In the present study, hamster relaxin was purified from placentas and characterized in order to investigate its biochemical properties. Placentas from Days 14 and 15 of gestation were homogenized in 0.26 N HCl-62.5% acetone containing protease inhibitors. After centrifugation, soluble proteins were acetone precipitated. Soluble proteins were applied to a carboxymethyl cellulose ion-exchange column and bound proteins were eluted with 0.1 and 0.3 M NaCl. Western blot analysis detected 16.5-, 18.7-, and 36.0-kDa relaxin-immunoreactive (IR) proteins within the 0.1 M NaCl eluant and detected a 5.6-kDa relaxin-IR protein within the 0.3 M NaCl eluant. The 5.6-kDa protein was purified to homogeneity by gel filtration (Sephadex G-50), ion-exchange HPLC, and C18-HPLC. Reduction of the 5.6-kDa protein prior to electrophoresis resulted in a single band of lower molecular mass, suggesting that hamster relaxin consists of two chains of approximately equal molecular mass. Isoelectric point of the 5.6-kDa protein was 7.78. The 16.5- and 18.7-kDa IR proteins were copurified by gel filtration and ion-exchange HPLC. At least five isoelectric point variants were observed for the 16.5- and 18.7-kDa proteins. The N-terminal amino acid for the 5.6 and 18.7 relaxin-IR proteins was arginine, and subsequent cycles indicated an identical partial sequence that was consistent with that for relaxins from other species.

Bilateral lesions of the pelvic nerve (BLPN) result in dystocia, but the processes which control this effect are not fully understood. Plasma progesterone, relaxin, and luteinizing hormone (LH) concentrations were measured in blood samples taken in the morning (AM) and evening (PM) of Days 20-23 of gestation from rats with BLPN or sham neurectomy. Ten of 11 sham-operated control animals delivered their entire litters by Day 23 of gestation, but animals with BLPN did not complete parturition by Day 23 when they were sacrificed. Progesterone concentrations were greater in rats with BLPN than in sham-operated rats on Day 20 PM and Day 21 AM, but hormone concentrations declined to minimal values by Day 22 in both groups. Relaxin concentrations were greater in rats with BLPN than in sham-operated rats on Day 21 PM. Thereafter, relaxin concentrations decreased to reach minimum values on Day 23 in both groups. LH concentrations were low throughout the period of study in rats with BLPN; however, a postpartum LH surge was detected in all sham-operated animals. Data from this study indicate that the pelvic nerve does not control parturition by modulating serum relaxin and progesterone concentrations; however, these data suggest that impulses carried by the pelvic nerve influence ovarian secretion of these hormones. In addition, these data indicate that the pelvic nerve transmits stimuli from the cervix to the hypothalamus to facilitate the postpartum LH surge.

Relaxin immunoreactivity was previously demonstrated in three cell types within the hamster placenta; fetal primary and secondary giant trophoblast cells (GTCs) and maternal endometrial granulocytes. The objectives of the present research were to examine the ultrastructure of the GTCs and identify the intracellular relaxin storage site. Primary GTCs, first present on day 8 of gestation, were characterized by numerous polyribosomes and large heterogeneous cytoplasmic inclusions suggesting phagocytic activity. Primary and secondary GTCs from days 10, 14, and 15 of gestation contained numerous polyribosomes, mitochondria with tubular cristae, and extensive Golgi complex, and abundant rough endoplasmic reticulum, all characteristics of a cell actively involved in protein synthesis. Membrane-bound secretory granules were not present. Relaxin was immunolocalized within the Golgi complex of primary and secondary GTCs using the avidin-biotin-peroxidase method. Following differential centrifugation of hamster placental homogenates and radioimmunoassay (RIA) of subcellular fractions, the majority of relaxin immunoactivity was detected in the postmicrosomal fraction; however, the majority of relaxin immunoactivity from similarly treated pig corpora lutea was present in the mitochondrial/granule fraction. These data indicate that hamster placental relaxin is not stored in membrane-bound secretory granules but is contained within the extensive Golgi complex of the GTC.

Two hormones with lactogenic activity are produced by the hamster placenta during the second half of pregnancy. One of these hormones, hamster placental lactogen II (haPL-II), has been well characterized; however, its cellular source is not known. In the present study, haPL-II was localized in placental tissues using a specific antibody and the avidin-biotin-peroxidase immunohistochemical technique. Because relaxin has been localized in the hamster placenta, it was of interest to determine if haPL-II and relaxin are localized in the same cells. haPL-II immunoactivity was observed in primary and secondary giant trophoblast cells of the placenta on Days 12, 14, and 15 of pregnancy. On Day 15 positive staining was also observed in large cells located within mesometrial arteries and in eosinophilic bodies associated with degenerating sheathed arteries of the decidua basalis. haPL-II-positive staining was not observed in placentae from Days 8 or 10 of pregnancy. On Day 14, haPL-II was colocalized with relaxin in 75% of the giant trophoblast cells observed. Therefore, it is probable that these hormones are synthesized and secreted by the same cell.

Bilateral section of either the sensory or motor branch of the pelvic nerve or pudendal nerve was performed in rats on days 8-10 of pregnancy, and the effects on delivery were observed. Bilateral resection of the sensory branch of the pelvic nerve reduced the number of live pups per litter, and increased the number of stillbirths and the number of fetuses retained in utero per litter at day 24. Sectioning motor components of the pelvic nerve, or both motor and sensory components of the pudendal nerve, had no effects on delivery in rats. We conclude that of the peripheral nerves evaluated in this study, only the sensory branch of the pelvic nerve is required for normal vaginal delivery in this species.

Uterine adrenergic and cholinesterase (AChE)-positive innervation of the sheep uterus during anestrus and at 4 stages of pregnancy were examined by histochemical methods. In addition, uterine and cervical myometrium concentrations of norepinephrine (NE) and dopamine (DA) were determined using high-performance liquid chromatography. During anestrus, adrenergic and AChE-positive nerve fibers in the uterine myometrium and endometrium were primarily associated with the vasculature. Innervation of myometrial smooth muscle was almost exclusively by adrenergic fibers. In the endometrium, fibers of both types were observed closely associated with endometrial glands, and adrenergic fibers were observed in the connective tissue beneath the luminal epithelium. Density of uterine innervation decreased by day 65 of pregnancy with an additional decrease by day 105. Myometrial NE concentrations were higher in the cervix than the uterus. Uterine NE concentrations generally were not affected by pregnancy. Although cervical NE per gram of tissue decreased during pregnancy, this effect of pregnancy was not detected when NE was expressed per microgram of DNA. Myometrial DA concentrations were higher in uterine segments than in the cervix. DA concentrations decreased during pregnancy in all tissues except the posterior uterine segment. The DA to NE ratio in the uterus was greater than that for the cervix and was not generally affected by the stage of pregnancy. These results demonstrate that cholinergic and adrenergic nerves supply the sheep uterus. Decreasing fiber density during pregnancy suggests that a majority of the innervation to the sheep uterus is supplied by 'short' nerve fibers whose activity is regulated by steroids of pregnancy. The possible role of DA as a neurotransmitter in the sheep uterus is discussed.

Uteroferrin, the iron-containing, progesterone-induced phosphatase of the porcine uterus, is a glycoprotein carrying a single oligosaccharide chain. Most of the uteroferrin isolated from either uterine secretions or allantoic fluid has endoglycosidase H-sensitive carbohydrate chains with either five or six mannose residues. As determined by 1H-NMR spectroscopy, the Man6 oligosaccharide has the following structure. (Formula: see text) The Man5 species lacks the terminal alpha 1,2-linked residue. Uteroferrin is transported across the pig placenta and has been proposed to be involved in iron transfer to the fetus (see Buhi, W. C., Ducsay, C. A., Bazer, F. W., and Roberts, R. M. (1982) J. Biol. Chem. 257, 1712-1721). Injection of 125I-labeled uteroferrin into the umbilical vein of midpregnant fetuses resulted in incorporation of label into the liver, the major site of fetal erythropoiesis. Light and electron microscope autoradiography revealed that the primary sites of uteroferrin uptake were the reticuloendothelial cells lining the liver sinusoids. Reticuloendothelial cells isolated from either fetal pig or adult rat livers were shown to accumulate uteroferrin when cultured in vitro. Uptake was inhibited by yeast mannan and by glycopeptides isolated from either ovalbumin or uteroferrin. Rat cells did not accumulate uteroferrin whose high mannose chains had been removed using endoglycosidase H. Moreover, the K uptake values (3 X 10(-7) M), specific competition by D-mannose and L-fucose bovine serum albumin, and inhibition by EDTA are consistent with an uptake mechanism involving a receptor for high-mannose oligosaccharides on the liver sinusoidal cells. It is suggested that one function of this receptor in the fetal pig is to remove maternally derived uterine glycoproteins from the fetal circulation. In the case of uteroferrin this process provides iron to the fetal liver.

Endometrial gland cells in uteri from late-pregnant guinea pigs (day 60 to parturition) resembled typical protein-secreting cells. Extensive rough endoplasmic reticulum and well-developed Golgi complexes were evident. The most striking features of endometrial gland cells were accumulations of large (approximately equal to 0.5 micron in diameter), dense, membrane-bounded granules. The granules were located in the supranuclear region of the cell and frequently occurred in close proximity to the plasma membrane adjacent to the lumen of the gland. Thin sections of endometrial gland cells treated with relaxin antiserum and either colloidal gold-protein A or colloidal gold-goat antirabbit IgG demonstrated that the granules contained relaxin. These studies provide additional evidence that the uterus of the guinea pig produces relaxin and support the hypothesis that uterin relaxin may play an important role in pregnancy and parturition in the guinea pig.

Uteri and ovaries from cycling, pregnant, and lactating guinea pigs were studied for immunolocalization of relaxin with the light microscope. Endometrial gland cells (EGC) from the same group of animals were examined in the electron microscope for the presence of secretory granules. Those EGC that exhibited high numbers of granules were stained either for relaxin with the protein A colloidal gold method or for carbohydrate with the thiocarbohydrazide technique. Relaxin was found in EGC from middle and late pregnant animals but was not detected in ovaries or uteri from cycling animals. While cytoplasmic granules were noted in most EGC from cycling animals examined, the number of granules was greatest in uteri from estrus and proestrus animals. Granules in EGC from estrus animals contained a carbohydrate-rich material but did not contain relaxin. Endometrial gland cells from animals in early to middle stages of pregnancy (days 15 and 30) contained limited numbers of granules, almost all of which contained carbohydrate. At day 45 of pregnancy, EGC containing many granules were noted. The majority of granules contained relaxin; however, a significant number of EGC contained carbohydrate-rich granules. Infrequently, EGC were noted that contained two populations of granules, and these two populations were assumed to be made up of relaxin-containing and carbohydrate-rich granules. EGC from animals on day 60 of pregnancy typically contained granules, and the majority of these contained relaxin. Carbohydrate-rich granules were observed in EGC of the day 60 animals but were smaller in diameter and were noted in much lower numbers than the relaxin-containing granules. Endometrial gland cells from lactating animals infrequently contained granules. These studies are consistent with the hypothesis that the uterus is the primary source of relaxin in the guinea pig and that relaxin plays an important role in pregnancy and parturition of this species. The observations implicate endometrial glands and their products in the physiology of the cycling animal as well as the pregnant and parturient animal.

Uteroferrin is a purple iron-containing acid phosphatase secreted by the porcine uterus under the influence of the hormone, progesterone. It is synthesized by the glandular epithelial cells of the uterine endometrium and during pregnancy is taken up by specialized structures (areolae) opposite each uterine gland. Uteroferrin is then released into the fetal circulation and cleared by the liver or fetal kidney. A major role in iron transport to the fetus has been proposed. Uteroferrin, as purified from uterine secretions of pigs, possesses mainly high mannose (predominantly Man5 and Man6) chains. These oligosaccharide chains of uteroferrin appear to be responsible for its binding and uptake by reticuloendothelial cells of the fetal liver which is the major site of erythropoiesis of the fetus. Uteroferrin, although implicated in transplantal iron transport, also possesses many of the properties of a lysosomal enzyme and, when newly synthesized, carries the so-called lysosomal recognition marker, mannose 6-phosphate. The phosphate group is masked by a covering N-acetylglucosamine residue, a feature which may account for its secretion rather than retention within lysosomes. Evidence is also presented that the oligosaccharide chains of newly synthesized uteroferrin are larger than those of the mature form and are trimmed after secretion. The phosphate group is also removed. It is not clear whether uteroferrin carbohydrate is implicated in the movement of the glycoprotein across the placenta as well as its uptake by the fetal liver.

These studies were designed to determine the tissue source of ovine relaxin and to determine the feasibility of using the pregnant ewe for study of relaxin production and secretion. On Day 4 of gestation, ewes were laparotomized, the nonpregnant uterine horn was ligated, and the ovary not containing the corpus luteum was removed. During a second surgery at Day 45 (n = 8) or 140 (n = 9) of gestation, 10-ml blood samples were drawn from a uterine artery, the ovarian vein, and veins draining the pregnant and nonpregnant uterine horns. Endometrial, placental, and luteal tissues were obtained for immunocytochemistry and extraction. Relaxin was detected by a heterologous porcine radioimmunoassay (RIA) in 3 of 54 serum samples (701.3 +/- 25.4 pg/ml, mean +/- SEM). Relaxin was not detected in crude tissue extracts, but low quantities were detected by RIA following Sephadex G-50 column chromatography of tissue extracts. Total relaxin activity for all tissues was equivalent to 0.57 +/- 0.13 ng of porcine relaxin/g tissue (w.w.). Relaxin was not detected immunocytochemically by light or electron microscopy. These data indicate that low quantities of relaxin are present in tissues and sera of pregnant ewes.

The objective of this study was to determine the tissue source of relaxin in pregnant hamsters by immunocytochemical techniques. Ovarian, uterine, and placental tissues were recovered from hamsters on Days 8, 10, 12, 14, and 15 of gestation and processed for light microscopy. Relaxin immunoreactivity was localized in tissue sections by the avidin-biotin-peroxidase technique using antiserum to porcine relaxin. On Day 8 of gestation, relaxin immunoreactivity was localized in primary giant trophoblast cells (GTC-1s) adjacent to the uterine decidua. On Day 10, relaxin immunoreactivity was localized in GTC-1s, secondary giant trophoblast cells (GTC-2s) adjacent to the ectoplacental cone, and endometrial granulocytes in the wall of sheathed arteries. On Day 12, relaxin immunoreactivity was observed primarily in GTC-2s interspersed among cells of the placental trophospongium but not in cells of the placental labyrinth. The intensity of staining and number of relaxin immunoreactive GTCs increased between Days 12 and 14 but was decreased by Day 15 PM. Relaxin was not localized in uterine glands or corpora lutea. These observations suggest that the placenta is the tissue source of relaxin in pregnant hamsters.

Placental transport of uteroferrin (Uf), the progesterone-induced iron transport glycoprotein, and its distribution within the fetus were investigated by the peroxidase-antiperoxidase bridge (PAP) technique. In Experiment 1, Uf was localized in endometrial and placental tissues taken from gilts on Days 60, 75, 90 and 105 of pregnancy. Uteroferrin was observed within cells of the endometrial glands and surface epithelium adjacent to placental areolae but not in endometrial surface epithelium between areolae. Heavy staining for Uf was observed in cells of the areolae and was associated with both supra- and infranuclear cytoplasmic vesicles. Vesicles located within the infranuclear cytoplasm were occasionally observed to be releasing their contents into capillaries surrounding the areolae. In Experiment 2, Uf was measured by radioimmunoassay in blood samples taken from the umbilical vein and artery of fetuses on Day 75 of pregnancy. Uteroferrin concentrations were greater (P less than 0.07) in umbilical vein blood (79.8 +/- 13.1 ng/ml) than in umbilical artery blood (43.9 +/- 13.1 ng/ml). Uteroferrin binding by Day 75 fetal liver membranes was examined in Experiment 3. Binding of 125I-Uf increased linearly with increasing quantities of membrane protein and binding of 125I-Uf was competitively inhibited by adding unlabeled Uf to the assay. In Experiment 4, urine samples were taken from Day 75 fetuses and assayed for beta-mercaptoethanol activated acid phosphatase activity which is indicative of Uf. In addition, urine proteins were analyzed for Uf by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and Ouchterlony double immunodiffusion (ID). Acid phosphatase specific activity was 3.37 +/- 1.83 mumol substrate hydrolyzed/10 min per mg protein. Uteroferrin was detected in two of three urine samples by 2D-PAGE and in three of eight samples by ID. In Experiment 5, tissue distribution of Uf was determined by the PAP technique in liver and kidney tissue taken from fetuses on Day 75 of pregnancy. Staining for Uf was observed in collecting ducts and proximal tubules of kidney tissue, but staining was not observed in liver tissue. These results indicate that Uf is transported by the areolae into the chorioallantoic capillaries and to the fetus by the umbilical vein. Within the fetus Uf is either bound by the liver, probably to supply iron for hematopoiesis, or cleared by the kidney and transported within the urine to the allantoic sac to serve as a temporary iron storage reservoir.