Biosynthesis Of Secretory Proteins Research Articles

Role of the unfolded protein response in determining the fate of tumor cells and the promise of multi-targeted therapies.

Although there have been advances in our understanding of carcinogenesis and development of new treatments, cancer remains a common cause of death. Many regulatory pathways are incompletely understood in cancer development and progression, with a prime example being those related to the endoplasmic reticulum (ER). The pathological sequelae that arise from disruption of ER homeostasis are not well defined. The ER is an organelle that is responsible for secretory protein biosynthesis and the quality control of protein folding. The ER triggers an unfolded protein response (UPR) when misfolded proteins accumulate, and while the UPR acts to restore protein folding and ER homeostasis, this response can work as a switch to determine the death or survival of cells. The treatment of cancer with agents that target the UPR has shown promising outcomes. The UPR has wide crosstalk with other signaling pathways. Multi-targeted cancer therapies which target the intersections within signaling networks have shown synergistic tumoricidal effects. In the present review, the basic cellular and signaling pathways of the ER and UPR are introduced; then the crosstalk between the ER and other signaling pathways is summarized; and ultimately, the evidence that the UPR is a potential target for cancer therapy is discussed. Regulation of the UPR downstream signaling is a common therapeutic target for different tumor types. Tumoricidal effects achieved from modulating the UPR downstream signaling could be enhanced by phosphodiesterase 5 (PDE5) inhibitors. Largely untapped by Western medicine for cancer therapies are Chinese herbal medicines. This review explores and discusses the value of some Chinese herbal extracts as PDE5 inhibitors.

Endoplasmic Reticulum Redox State Is Not Perturbed by Pharmacological or Pathological Endoplasmic Reticulum Stress in Live Pancreatic β-Cells

Accumulation of unfolded, misfolded and aggregated proteins in the endoplasmic reticulum (ER) causes ER stress. ER stress can result from physiological situations such as acute increases in secretory protein biosynthesis or pathological conditions that perturb ER homeostasis such as alterations in the ER redox state. Here we monitored ER redox together with transcriptional output of the Unfolded Protein Response (UPR) in INS-1 insulinoma cells stably expressing eroGFP (ER-redox-sensor) and mCherry protein driven by a GRP78 promoter (UPR-sensor). Live cell imaging, flow cytometry and biochemical characterization were used to examine these parameters in response to various conditions known to induce ER stress. As expected, treatment of the cells with the reducing agent dithiothreitol caused a decrease in the oxidation state of the ER accompanied by an increase in XBP-1 splicing. Unexpectedly however, other treatments including tunicamycin, thapsigargin, DL-homocysteine, elevated free fatty acids or high glucose had essentially no influence on the ER redox state, despite inducing ER stress. Comparable results were obtained with dispersed rat islet cells expressing eroGFP. Thus, unlike in yeast cells, ER stress in pancreatic β-cells is not associated with a more reducing ER environment.

COP-binding sites in p24δ 2 are necessary for proper secretory cargo biosynthesis

The p24 family is thought to be somehow involved in endoplasmic reticulum-to-Golgi protein transport, and its members are major constituents of transport vesicles and bind to the vesicle coat protein complexes COPI and COPII. A subset of the p24 proteins (p24α 3, -β 1, -γ 3 and -δ 2) is upregulated when Xenopus laevis intermediate pituitary melanotrope cells are physiologically activated to produce vast amounts of their major secretory cargo, the prohormone proopiomelanocortin (POMC). To investigate the role of the COP-binding motifs of p24 proteins in POMC biosynthesis, we here generated and analysed Xenopus with stable, melanotrope cell-specific transgene expression of p24δ 2-GFP mutated in its COPI- or COPII-binding motif. In contrast to what has been found previously for wild-type (wt) p24δ 2-GFP, the p24δ 2 mutations prevented the Golgi localisation of the transgene products and caused a reduced rate of POMC cleavage, but did not lead to a reduction of the endogenous p24 proteins nor to aberrations in POMC glycosylation and sulphation. We conclude that p24δ 2 requires the presence of the COPI- and COPII-binding sites to allow proper POMC processing. Thus, the p24 proteins fulfil their role in secretory protein biosynthesis via COPI- or COPII-coated transport vesicles.

Ribosome Binding of a Single Copy of the SecY Complex: Implications for Protein Translocation

The SecY complex associates with the ribosome to form a protein translocation channel in the bacterial plasma membrane. We have used cryo-electron microscopy and quantitative mass spectrometry to show that a nontranslating E. coli ribosome binds to a single SecY complex. The crystal structure of an archaeal SecY complex was then docked into the electron density maps. In the resulting model, two cytoplasmic loops of SecY extend into the exit tunnel near proteins L23, L29, and L24. The loop between transmembrane helices 8 and 9 interacts with helices H59 and H50 in the large subunit RNA, while the 6/7 loop interacts with H7. We also show that point mutations of basic residues within either loop abolish ribosome binding. We suggest that SecY binds to this primary site on the ribosome and subsequently captures and translocates the nascent chain.

Insulin induces chaperone and CHOP gene expressions in adipocytes

Adipocyte secretes bioactive proteins called adipocytokines, and biosynthesis of secretory proteins requires molecular chaperones and folding enzymes in endoplasmic reticulum (ER). ER chaperones are known to be induced by unfolded protein response (UPR) and growth factors, however, it has not been determined how ER chaperones expression is regulated in adipocytes. Here we show that insulin treatment induced GRP78 and ERO1L mRNA levels in 3T3-L1 adipocytes. Insulin also upregulated CHOP mRNA levels, but did not induce phosphorylation of eIF2α. Pretreatment with insulin protected 3T3-L1 adipocytes against thapsigargin-mediated phosphorylation of eIF2α but did not against DTT-mediated one. In vivo mice study showed that GRP78 and CHOP expressions were regulated by feeding conditions. These results suggest that insulin signaling is important to induce mRNA expressions of GRP78 and CHOP, and may have a protective role against UPR.

Catestatin, the catecholamine release-inhibitory peptide fragment of chromogranin A: Naturally occurring human variants displaying different potencies for multiple nicotinic cholinergic responses in chromaffin cells

Resequencing of the human chromogranin A gene identified three human variants of catestatin: Gly364Ser, Pro370Leu, and Arg374Gln. Naturally occurring variants were tested for ability to inhibit four nicotinic processes. The rank order of potency for inhibition of catecholamine secretion was Pro370Leu> wild type> Gly364Ser> Arg374Gln. Decrease in potency was paralleled by decline in Hill slope, suggesting that negative cooperativity at ascending dose might underlie loss of potency. Each variant acted as a nicotinic antagonist. Potency to inhibit secretion paralleled inhibition of agonist-triggered 22Na+ uptake (r=0.986 and blockade of secretion was non-competitive with agonist. Variants also inhibited desensitization of secretion after prior agonist exposure, and stimulation of secretory protein biosynthesis by agonist. Rank order of variant inhibitory potency for all four nicotinic processes was identical (Pro370Leu > wild type > Gly364Ser > Arg374Gln), suggesting mediation by similar combinations of receptor alpha/beta subunits, and that crucial catestatin residues are likely to be identical across the four processes. When catestatin variants were mixed in likely heterozygotic (1:1 molar ratio) combinations, the inhibitory curve was left-shifted onto that of the more potent variant in the combination, suggesting not only phenotypic dominance. The results from four nicotinic processes have quantitative implications for inter-individual variations in human nicotinic signaling. Clinical Pharmacology & Therapeutics (2005) 77, P6–P6; doi: 10.1016/j.clpt.2004.11.027

The catecholamine release-inhibitory "catestatin" fragment of chromogranin a: naturally occurring human variants with different potencies for multiple chromaffin cell nicotinic cholinergic responses.

The catestatin fragment of chromogranin A is an endogenous inhibitor of nicotinic cholinergic transmission, functioning in negative feedback control of catecholamine secretion. We explored naturally occurring polymorphisms in the amino acid sequence of catestatin. Three human variants were identified: Gly364Ser, Pro370Leu, and Arg374Gln. Variants were tested for ability to inhibit four nicotinic processes. The rank order of potency for inhibition of catecholamine secretion was Pro370Leu > wild type > Gly364Ser > Arg374Gln. Decrease in potency was paralleled by decline in Hill slope, suggesting that negative cooperativity at ascending dose might underlie loss of potency. Several lines of evidence indicated that each variant acted as a nicotinic antagonist: potency to inhibit secretion paralleled inhibition of agonist-triggered (22)Na(+) uptake (r = 0.986); variants inhibited secretion with similar potency when triggered by several nicotinic agonists, though not by agents using other secretory pathways or bypassing the nicotinic receptor; and blockade of secretion was noncompetitive with agonist. Variants also inhibited desensitization of secretion after prior agonist exposure and stimulation of secretory protein biosynthesis by agonist. Rank order of variant inhibitory potency for all four nicotinic processes was identical (Pro370Leu > wild type > Gly364Ser > Arg374Gln), suggesting mediation by similar combinations of receptor alpha/beta subunits and that crucial catestatin residues are likely to be identical across the four processes. When catestatin variants were mixed in likely heterozygotic (1:1 M ratio) combinations, the inhibitory curve was left-shifted onto that of the more potent variant in the combination, suggesting phenotypic dominance. The results have quantitative implications for interindividual variations in human nicotinic signaling.

Gene microarray analysis reveals interleukin-5–dependent transcriptional targets in mouse bone marrow

AbstractInterleukin-5 (IL-5) is a hematopoietic differentiation factor that promotes the development of mature eosinophils from progenitors in bone marrow. We present a multifactorial microarray study documenting the transcriptional events in bone marrow of wild-type and IL-5–deficient mice at baseline and in response to infection with Schistosoma mansoni. The microarray data were analyzed by a 4-way subtractive algorithm that eliminated confounding non-IL-5–related sequelae of schistosome infection as well as alterations in gene expression among uninfected mice. Among the most prominent findings, we observed 7- to 40-fold increased expression of transcripts encoding the classic eosinophil granule proteins (eosinophil peroxidase, major basic protein, the ribonucleases) together with arachidonate-15-lipoxygenase and protease inhibitor plasminogen activator inhibitor 2 (PAI-2), in the IL-5–producing, infected wild-type mice only. This was accompanied by increased transcription of genes involved in secretory protein biosynthesis and granule-vesicle formation. Interestingly, we did not detect increased expression of genes encoding eosinophil-related chemokine receptors (CCR1, CCR3) or members of the GATA or CCAAT/enhancer binding protein (C/EBP) transcription factor families. These data suggest that the IL-5–responsive progenitors in the mouse bone marrow are already significantly committed to the eosinophil lineage and that IL-5 promotes differentiation of these committed progenitors into cells with recognizable and characteristic cytoplasmic granules and granule proteins.

Comparative ultrastructural study of endoplasmic reticulum in colorectal carcinoma cell lines with different degrees of differentiation.

The endoplasmic reticulum (ER) consists of a complex system of tubules, lamellae , and flattened vesicles, and has a variety of morphologies in different cells. It is believed to play a central role in the biosynthesis of cholesterol, phosph olipids, steroids, prostaglandins, membrane and secretory proteins[1]. Cancer cells have different functions and ultrastructure from their original cells[2-4]. The studies on ER membrane system of cancer cells are of great significance in understanding their malignant behavior. In the present work, the ultrastructural characteristics of ER in human colorectal carcinoma cell lines with different differentiation degrees were investigated.

The role of coat proteins in the biosynthesis of secretory proteins

The biosynthesis of secretory proteins requires vesicle-mediated transport between the organelles of the secretory pathway. Biochemical and genetic analysis of the secretory pathway has identified two non-clathrin coats—COPI and COPII — that drive the formation of vesicles that mediate transport between the endoplasmic reticulum and the Golgi apparatus, and through the compartments of the Golgi. Recently, a molecular description of the subunits of these coats and the development of biochemical reagents to study their function has yielded new information on how these proteins share the task of organizing vesicle traffic early in the secretory pathway.

Temperature and androgens regulate the biosynthesis of secretory proteins from rabbit cauda epididymidis.

We have compared the biosynthesis of secretory proteins in rabbit cauda epididymidis maintained for 15 days at abdominal temperature with that of the scrotal cauda. Explants from both situations were incubated in vitro in the presence of [35S] methionine, and the labelled proteins released into the incubation medium were analyzed by polyacrylamide gel electrophoresis. Body temperature specifically inhibited the synthesis of at least two polypeptides of 43 kDa and 21 kDa (designated EP21), whereas the synthesis of polypeptides of 80, 39, 31, and 24 kDa was increased. These changes resembled those produced by castration, but androgen treatment was not able to reverse the effect of body temperature. To confirm these observations, poly(A)+ RNA from the scrotal and the abdominal cauda respectively, was translated in vitro and the synthesized products were immunoprecipitated with an antibody against EP21 polypeptide. Both castration and body temperature strongly decreased the concentration of EP21 mRNA. In vivo testosterone administration restored the content of EP21 mRNA in cauda from castrated animals, but not in cauda maintained at body temperature. The changes observed might be related to the adverse effect of body temperature on sperm storage in the cauda epididymidis.

Enkephalin biosynthesis is coupled to secretory activity via transcription of the proenkephalin A gene

The molecular mechanisms regulating neuropeptide and secretory protein biosynthesis in neuroendocrine cells were examined using the prototype neuropeptide and secretory proteins enkephalin and chromogranin A (CGA). Treatment with the secretogogue nicotine results in the calcium-dependent secretion of enkephalin peptides from bovine chromaffin cells in primary culture and a concomitant increase in enkephalin peptide biosynthesis. Both secretion and biosynthesis are also stimulated by cell depolarization with elevated potassium. Elevation of intracellular cyclic AMP, on the other hand, results in stimulation of enkephalin biosynthesis and long-term, but not acute, secretion of enkephalin peptides. Coupling of enkephalin biosynthesis to calcium influx occurs at the level of transcription of the enkephalin gene. Thus, potassium depolarization causes a calcium-dependent elevation of enkephalin mRNA which is preceded by an increase in the rate of transcription of the enkephalin gene in the chromaffin cell. The accumulation of enkephalin message or peptide by potassium depolarization or treatment with nicotine is prevented by D600 or hexamethonium respectively, added 1 h after addition of nicotine or KCl and following acute release, suggesting that calcium acts as a continuous rather than triggering stimulus for enkephalin biosynthesis. Sequence analysis of the bovine enkephalin promoter identified sequence conservation of three enhancers previously reported in the human gene which are required for regulation of the gene by calcium, cAMP, and phorbol ester in vitro. In contrast to the regulation of the enkephalin system, no increase in either CGA or CGB mRNA or gene transcription attended depolarization-induced secretion from chromaffin cells. Since enkephalin and CGA are co-stored in and co-released from the same secretory vesicles in these cells, the results imply that a surplus of CGA is constitutively synthesized in chromaffin cells such that compensatory up-regulation during changes in the secretory state of the cell, such as occurs for enkephalin, is not required for the secretory proteins.

Protein transport across the endoplasmic reticulum membrane: facts, models, mysteries

The first step in the biosynthesis of secretory proteins, plasma membrane proteins, and many other proteins in a eukaryotic cell involves the transport of at least portions of the polypeptides across the endoplasmic reticulum (ER) membrane. Parts of the polypeptide chains serve as signals that direct the translocation across and the integration into the ER membrane and also determine the orientation of membrane proteins. The transport process itself may be divided into two phases: an initiation or targeting cycle, which is fairly well understood, and the actual transfer of the polypeptide chain through the membrane, the mechanism of which is still mysterious. The initiation cycle generally involves the function of the signal recognition particle (SRP) which binds to signal sequences through its 54-kDa polypeptide component, and of the SRP-receptor (docking protein) in the ER membrane whose function is dependent on GTP. The membrane transfer of the polypeptide chain appears to involve a translocation complex consisting of several membrane proteins, one of which is the signal sequence receptor protein (SSR).

Modulations of the epithelial phenotype and functional activity of cultured bovine tracheal gland cells: dependence on the culture medium and passage number

Bovine tracheal gland (BTG) cells in culture show an epithelial-fibroblastoid transition after several passages. To investigate these BTG cell phenotype changes, we studied the effects of both the culture medium and passage number on the expression of epithelial cytoskeletal proteins and glandular serous cell markers. We also analyzed the intracellular cAMP level in the basal state and after adrenergic stimulation. Three culture media were used: 1) serum-free defined medium (SFDM); 2) medium supplemented with 2% Ultroser G; and 3) medium supplemented with 10% fetal calf serum (FCS). Using immunofluorescence microscopy, we showed that, in the first 4 passages whatever the culture conditions, BTG cells expressed immunoreactivities to cytokeratin filaments and desmoplakins I and II, whereas vimentin filaments were not detected. After four passages, BTG cells cultured in 10% FCS or 2% Ultroser G became progressively fibroblastoid and showed immunoreactivities to both vimentin and cytokeratin intermediate filaments. No immunoreactivity to vimentin filaments was observed on BTG cells cultured in a SFDM. Using biochemical analysis, we showed that basal levels of cAMP in cultured BTG cells and lysozyme secretion by these cells vary according to the culture medium and passage number. It was higher in BTG cells cultured in a SFDM compared to that recovered from cells cultured in medium supplemented with Ultroser G or FCS. Whatever the culture medium, BTG cells responded to stimulation by isoproterenol. However, the results of stimulation in a SFDM were higher than in Ultroser G or FCS supplemented medium. We conclude that the BTG epithelial cell organization and the regulation of biosynthesis of secretory proteins by these cells in culture depend on both the culture medium and passage number.(ABSTRACT TRUNCATED AT 250 WORDS)

Defective terminal differentiation in squamous cell carcinomas and a hypothesis for oral cancer

Differentiation in malignant tumors is inversely related to progression, invasion and metastasis and is assessed on the basis of a marker, usually a secreted protein such as keratin. Recently, several ultrastructurally recognizable markers of differentiation were scored in breast tumors and the inadequacy in evaluating differentiation on the basis of a single marker was pointed out. The present investigations on squamous cell carcinomas of the tongue, buccal mucosa and nasopharynx (total of 15 samles), erythroplakia (one) and leukoplakia (3) have revealed a structural pathway underlying defective terminal differentiation in malignant cells.There are two types of protein synthesis in cells. One, on free cytoplasmic polysomes where enzymes for metabolic reactions and cytoskeletal proteins are thought to be synthesised. The second concerns biosynthesis of secretory proteins, membranes and lysosomal enzymes and takes place in the endomembrane system (EMS). The EMS is constituted by the nuclear envelope, rough endoplasmic reticulum (RER), Golgi apparatus, transport vesicles and lysosomes.

Secretory Protein Biosynthesis in Snail Hemocytes: In vitro Modulation by Larval Schistosome Excretory-Secretory Products

Circulating hemocytes of the snail, Biomphalaria glabrata, synthesize and secrete a variety of polypeptides when maintained in vitro in serum-free medium containing [35S] methionine. SDS-PAGE/fluorographic analysis of supernatants from resistant snail (10-R2-OK strain) hemocyte cultures revealed the presence of numerous labeled polypeptides ranging in Mr from 220 to 14 kDa. Most of these same proteins were also produced by hemocytes of a susceptible B. glabrata strain (M-line), but the overall rate of secretory protein synthesis was reduced from that of resistant snail cells. In addition, excretory-secretory (ES) products contained in supernatants from Schistosoma mansoni miracidial transformation and 1-day primary sporocyst cultures stimulated increases in the synthesis of various polypeptides. Particularly striking was a 3-fold increase in the synthesis of a 66-kDa secretory polypeptide by hemocytes of both snail strains, and a concomitant increase in M-line hemocytes and decrease in 10-R2-OK cells of a 63-kDa polypeptide. Overall, however, the level of ES product-induced secretory protein synthesis was greater in 10-R2-OK snail hemocytes than in those of the M-line strain. Exposure of a nonhemocytic B. glabrata cell line to parasite culture supernatants had no stimulatory/inhibitory effect on labeled protein ouput, suggesting that the observed hemocyte response may be snail cell-type specific. Finally, the larval ES components responsible for modulating hemocyte protein metabolism are mainly concentrated in a heat-stable fraction composed of molecules of greater than 30 kDa. However, the loss of the ability of heated parasite products to stimulate synthesis of certain hemocyte proteins and the presence of minor stimulating activity in a low molecular weight fraction (less than 10 kDa) implies the possible existence of multiple larval components affecting formation of specific hemocyte secretory polypeptides. It is concluded that snail hemocytes are capable of in vitro synthesis and secretion of a variety of methionine-containing polypeptides, and that ES products of early larval schistosomes can modulate (i.e., stimulate or inhibit) this metabolic process. A differential response of susceptible vs. resistant hemocytes to larval products suggests that the degree to which these cells can be metabolically activated may determine their cytotoxic effectiveness.

Alpha-Amylase biosynthesis: evidence for temporal sequence of NH2-terminal peptide cleavage and protein glycosylation.

During the biosynthesis of secretory proteins, an NH2-terminal peptide, referred to as a signal peptide, is cotranslationally cleaved off after the protein enters the cisternal space of the endoplasmic reticulum. It also has been reported that the core glycosylation reaction of some secretory and viral membrane glycoproteins occurs as a cotranslational event. However, despite a huge amount of work, no decisive answer has been given as to the temporal sequence of proteolytic cleavage of the signal peptide and glycosylation on the polysomes for any secretory or membrane glycoprotein. We show here that proteolytically processed and already glycosylated chains of rice seed alpha-amylase exist on the polysomes; furthermore, our results provide direct evidence that glycosylation is preceded by proteolytic processing during the biosynthesis of the alpha-amylase molecule.

Involvement of free ribosomes in the early stage of secretory protein biosynthesis in rat liver.

Nascent peptides on free and bound ribosomes prepared from rat liver were labeled and released with [3H]puromycin, and the amounts of the nascent peptides of two secretory proteins, serum albumin and transferrin, were determined by immunoprecipitation with specific antibodies. An appreciable amount of serum albumin nascent peptides was associated with free ribosomes, although most of them were carried by bound ribosomes. Serum albumin nascent peptides associated with free ribosomes were smaller in size than those carried by bound ribosomes. When the products of cell-free translation programmed by free and bound polysomes were immunoprecipitated with antibodies, the percentages of serum albumin to the total translation products were in good agreement with those obtained in the nascent peptides experiment. Cell-free translation of free polysomes produced serum albumin with a similar molecular weight to the authentic one. Although nascent peptides of transferrin were detected only on bound ribosomes, and not on free ribosomes, the transferrin antibody immunoprecipitated 0.2% of total translation products of free polysomes. These observations indicate that free polysomes have complete messenger RNA's and shorter growing nascent peptides of serum albumin and transferrin.

New Evidence for the Function of the Scrotum

Previous articleNext article No AccessNew Evidence for the Function of the ScrotumRichard E. HellerRichard E. Heller Search for more articles by this author PDFPDF PLUS Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinkedInRedditEmail SectionsMoreDetailsFiguresReferencesCited by Volume 2, Number 1Jan., 1929 Article DOIhttps://doi.org/10.1086/physzool.2.1.30151061 Views: 5Total views on this site Citations: 17Citations are reported from Crossref Journal History This article was published in Physiological Zoology (1928-1998), which is continued by Physiological and Biochemical Zoology (1999-present). PDF download Crossref reports the following articles citing this article:Mohamed A. Baky Fahmy Scrotal Functions and Missions, (Dec 2021): 49–55.https://doi.org/10.1007/978-3-030-83305-3_6Francisco Regalado, Pedro Esponda, Antonio Nieto Temperature and androgens regulate the biosynthesis of secretory proteins from rabbit cauda epididymidis, Molecular Reproduction and Development 36, no.44 (Dec 1993): 448–453.https://doi.org/10.1002/mrd.1080360407Dominique Marmor, Elisabeth Elefant, Caroline Dauchez, Charles Roux Semen analysis in Hodgkin's disease before the onset of treatment, Cancer 57, no.1010 (May 1986): 1986–1987.https://doi.org/10.1002/1097-0142(19860515)57:10<1986::AID-CNCR2820571017>3.0.CO;2-WJ. Michael Bedford, Gerald Zelikovsky Viability of Spermatozoa in the Human Ejaculate After Vasectomy, Fertility and Sterility 32, no.44 (Oct 1979): 460–463.https://doi.org/10.1016/S0015-0282(16)44305-0J. Michael Bedford Anatomical evidence for the epididymis as the prime mover in the evolution of the scrotum, American Journal of Anatomy 152, no.44 (Aug 1978): 483–507.https://doi.org/10.1002/aja.1001520404J. Michael Bedford Influence of abdominal temperature on epididymal function in the rat and rabbit, American Journal of Anatomy 152, no.44 (Aug 1978): 509–521.https://doi.org/10.1002/aja.1001520405H. E. Voss Biologie der Androgene, (Jan 1973): 327–528.https://doi.org/10.1007/978-3-642-80666-7_8G.L. Moench The longevity of the human spermatozoa, American Journal of Obstetrics and Gynecology 38, no.11 (Jul 1939): 153–155.https://doi.org/10.1016/S0002-9378(39)90498-9S. S. Munro The Effect of Testis Hormone on the Preservation of Sperm Life in the Vas Deferens of the Fowl, Journal of Experimental Biology 15, no.22 (Apr 1938): 186–196.https://doi.org/10.1242/jeb.15.2.186Samuel L. Leonard, James B. Hamilton Comparison and rate of testicular degeneration in rats after cryptorchidism and hypophysectomy, The Anatomical Record 68, no.44 (Jul 1937): 497–502.https://doi.org/10.1002/ar.1090680411William E. White The effect of hypophysectomy on the survival of spermatozoa in the male rat, The Anatomical Record 54, no.22 (Oct 1932): 253–273.https://doi.org/10.1002/ar.1090540212Martha C. Toothill, William C. Young The time consumed by spermatozoa in passing through the ductus epididymidis of the guinea-pig as determined by means of India-ink injections, The Anatomical Record 50, no.11 (Jul 1931): 95–107.https://doi.org/10.1002/ar.1090500109Fiorindo A. Simeone, William C. Young A Study of the Function of the Epididymis, Journal of Experimental Biology 8, no.22 (Apr 1931): 163–175.https://doi.org/10.1242/jeb.8.2.163R. M. C. Gunn, H. R. Seddon Testicular Grafts on Rams., Australian Veterinary Journal 6, no.44 (Dec 1930): 132–145.https://doi.org/10.1111/j.1751-0813.1930.tb15005.xR. M. C. Gunn, H. R. Seddon Testicular Grafts on Rams., Australian Veterinary Journal 6, no.33 (Sep 1930): 104–112.https://doi.org/10.1111/j.1751-0813.1930.tb14990.xR. M. C. Gunn Testicular Transplants in Goats., Australian Veterinary Journal 6, no.22 (Jun 1930): 56–65.https://doi.org/10.1111/j.1751-0813.1930.tb14970.xWilliam C. Young A study of the function of the epididymis. II. The importance of an aging process in sperm for the length of the period during which fertilizing capacity is retained by sperm isolated in the epididymis of the guinea-pig, Journal of Morphology 48, no.22 (Dec 1929): 475–491.https://doi.org/10.1002/jmor.1050480208