Bovine Testicular Tissue Research Articles

Localization and in silico-based functional analysis of miR-202 in bull testis.

Bull fertility is pivotal to the prosperity of the cattle industry worldwide. miR‐202 has been shown to be gonad specific and to have key roles in gonad function in different species. To further understand the involvement of miR‐202 in bull reproduction, this study aimed to establish its localization in bovine testicular tissue and to identify putative biological functions using bioinformatics approaches. We assessed the miR‐202 expression in paraffin‐embedded tissue samples collected form an abattoir using in situ hybridization. miR‐202 was present in Sertoli cells and in germ cells at different stages of development. Using available databases, a total of 466 predicted gene targets of miR‐202 were identified. Functional annotation revealed that miR‐202 target genes were mainly associated with protein modification and phosphorylation processes as well as longevity regulating pathway. Moreover, genes in the longevity regulating pathway mapped to PI3K/Akt/mTOR pathway which is involved in promoting proliferation of testicular cells and spermatogenesis. These findings suggest that miR‐202 plays important roles in regulating proliferation and viability of testicular cells including somatic and germ cells.

Detection of protamine 2 in bovine spermatozoa and testicles.

Sperm DNA integrity is crucial for transmission of genetic information to future generations and DNA damage can occur during chromatin packaging. Chromatin packaging involves the replacement of somatic nucleosomal histones by nuclear proteins called protamines. Protamine 1 (PRM1) is transcribed and translated in spermatids of all mammals; however, protamine 2 (PRM2) is transcribed in low levels in spermatids and it is not yet described in bull mature spermatozoa. The aim of this study was to assess gene and protein expression of PRM2 and corroborate gene and protein expression of PRM1 in bull spermatozoa and testis. For this purpose, absolute q-RT-PCR was performed to calculate the number of copies of PRM1 and PRM2 mRNAs in bovine epididymal spermatozoa and testicular tissue. Western blot and mass spectrometry were performed to identify PRM1 and PRM2 in samples of bovine epididymal spermatozoa. Samples of bovine testicular tissue were collected to identify PRM1 and PRM2 by immunohistochemistry. We evaluated that the number of PRM1 mRNA copies was about hundred times higher than PRM2 mRNA copies in sperm and testicular samples (p<0.0001). In addition, we estimated the PRM1: PRM2 ratio based on mRNA number of copies. In spermatozoa, the ratio was 1: 0.014, and in testicle, the ratio was 1: 0.009. We also evaluated the immunolocalization for PRM1 and PRM2 in bovine testis, and both proteins were detected in spermatids. Western blot and mass spectrometry in bovine epididymal spermatozoa confirmed these results. Our work identifies, for the first time, PRM2 in bovine epididymal spermatozoa and in testis. Further studies are still needed to understand the role of PRM2 on the chromatin of the spermatozoa and to verify how possible changes in PRM2 levels may influence the bull fertility.

Effects of five cryoprotectants on proliferation and differentiation-related gene expression of frozen-thawed bovine calf testicular tissue.

The cryopreservation of testicular tissue is a potential method for preserving male fertility. However, the effect of cryopreservation on bovine calf testicular tissue is scarce. This study investigated the effect of different cryoprotectants on bovine calf testicular tissue at the molecular level. Testicular tissue from ten immature bovine calves (6months) was collected after slaughter and cryopreserved in an extender containing different concentrations of the following five cryopreservation solutions (CP): bovine serum albumin (BSA) with 5% dimethyl sulfoxide (DMSO), trehalose with 5% DMSO, DMSO and glycerol and ethylene glycol (EG). After 7-day cryopreservation, the expression levels of three spermatogonial stem cell (SSC)-related genes, octamer-4 (OCT4), KIT ligand (MGF/SCF) and kit oncogene (C-KIT), were investigated by quantitative PCR (qPCR). The cell viability was highest for the tissues preserved with 30mg/ml BSA (77.82%±1.22) and 40mg/ml trehalose (74.23%±1.16) compared with other groups (p<0.05), and the level of expression of the three genes was highest with 30mg/ml BSA (p<0.05). Compared with other CPs, the 30mg/ml BSA and 40mg/ml trehalose have the better cryopreserve protection. The 30mg/ml BSA is the most viable media for the cryopreservation of testicular tissue from cattle.

Enrichment and invitro features of the putative gonocytes from cryopreserved testicular tissue of neonatal bulls.

Enrichment and propagation of gonocytes or spermatogonial stem cells (SSCs) from cryopreserved testicular tissue is essential to apply SSCs-related techniques in large domestic animals. We previously reported the cryopreservation of adult bovine testicular tissue. Here, we conducted the enrichment and culture of putative gonocytes from cryopreserved testicular tissues of post-natal 1-day-old bulls. The testicular structure was well maintained after freezing and thawing. Higher mRNA levels of gonocyte/SSCs markers (PLZF, GFRα1, and UCHL-1) than those of pluripotency genes (Oct4, Sox2, and Nanog) were detected in the frozen-thawed sex cords. GFRα1 was specifically detected in the membrane and cytoplasm of gonocytes by immunostaining. Differential plating provided 40-50% enrichment of putative gonocytes. They were single, paired-, aligned-cells, or grape cluster-like colonies in minimum essential medium (MEM) containing 2.5% FBS+2mM glutamine+100IU/mL penicillin-streptomycin+40μg/mL gentamycin+15mM HEPES+10mM β-mercaptoethanol+0.1mM non-essential amino acids+1mM sodium pyruvate. On day 3, gonocyte progeny increased and the contaminated somatic cells spread and concurrently divided slowly. On day 5, gonocyte progeny proliferated continuously and typical intercellular bridges formed by incomplete cytokinesis in paired-cells or aligned-cysts were observed. Immunochemically, they were still GFRα1 and PLZF positive. These cells expressed significantly higher gonocyte/SSCs marker mRNAs than pluripotency gene mRNAs, concomitant with a higher level of differentiated spermatogonia marker c-kit. With time, gonocyte progeny colonies appeared in varied sizes and expanded dramatically on day 7. After cultured for 9-10days, however, large colonies collapsed and dispersed as some single cells and small syncytial cysts. Together, MEM containing 10% dimethyl sulfoxide+2.5% newborn calf serum provides efficient cryoprotection for the testicular tissue from 1-day-old neonatal bulls. Putative gonocytes enriched from these nascent tissues present robust proliferation capacity, conserved gonocyte/SSCs markers, and SSCs-like invitro features.

Enrichment and culture of spermatogonia from cryopreserved adult bovine testis tissue

Propagation of bovine spermatogonial stem cells (SSCs) from the cryopreserved testicular tissue is essential for the application of SSCs-related techniques. To explore the appropriate conditions for in vitro culture of bovine spermatogonia (containing putative SSCs), Sertoli cell monolayer and serum concentration were set as two main control factors. Morphological examination showed that the intactness and structure of adult bovine testicular tissue were well maintained after cryopreservation. The enriched bovine spermatogonia were large round CD9 and promyelocytic leukemia zinc finger protein (PLZF) positive cells, with high nucleocytoplasmic ratios and multiple types including single, paired-, aligned-cells or grape cluster-like colonies in vitro. In Sertoli cell co-culture system, bovine spermatogonia attached quickly and proliferated obviously faster than those in the system without Sertoli cells. Serum-free media was no good for the attachment and proliferation of bovine spermatogonia. When 2.5%, 5% and 10% fetal bovine serum (FBS) was employed in the media, spermatogonia attached easily and divided quickly to form paired-, chained-cells or grape cluster-like colonies with comparable percentages in all groups. However, the contaminated somatic cells proliferated robustly in groups containing 5% and 10% FBS. Together, bovine spermatognia isolated from cryopreserved adult testis tissue express CD9 and PLZF, can survive and proliferate conspicuously in Sertoli cell co-culture system, and low serum provides an optimal condition for the survival and proliferation of bovine spermatogonia because of avoiding the rapid growth of testis somatic cells.

Effects of trehalose supplementation on cell viability and oxidative stress variables in frozen-thawed bovine calf testicular tissue

Trehalose is widely used for cryopreservation of various cells and tissues. Until now, the effect of trehalose supplementation on cell viability and antioxidant enzyme activity in frozen-thawed bovine calf testicular tissue remains unexplored. The objective of the present study was to compare the effect of varying doses of trehalose in cryomedia on cell viability and key antioxidant enzymes activities in frozen-thawed bovine calf testicular tissue. Bovine calf testicular tissue samples were collected and cryopreserved in the cryomedias containing varying doses (0, 5, 10, 15, 20 and 25%; v/v) of trehalose, respectively. Cell viability, total antioxidant capacity (T-AOC) activity, catalase (CAT) activity, superoxide dismutase (SOD) activity, glutathione (GSH) content and malondialdehyde (MDA) content were measured and analyzed. The results showed that cell viability, T-AOC activity, SOD activity, CAT activity and GSH content of frozen-thawed bovine calf testicular tissue was decreased compared with that of fresh group (P<0.05). MDA content in frozen-thawed bovine calf testicular tissue was significantly increased compared with that of fresh group (P<0.05). The cryomedia added 15% trehalose exhibited the greatest percentage of cell viability and antioxidant enzyme activity (SOD and CAT) among frozen-thawed groups (P<0.05). Meanwhile, GSH content was the lowest among frozen-thawed groups (P<0.05). However, there were no significance differences in MDA content among the groups added 10, 15 and 20% trehalose (P>0.05). In conclusion, the cryomedia added 15% trehalose reduced the oxidative stress and improved the cryoprotective effect of bovine calf testicular tissue. Further studies are required to obtain more concrete results on the determination of antioxidant capacity of trehalose in frozen-thawed bovine calf testicular tissue.

Splice variants and promoter methylation status of the Bovine Vasa Homology (Bvh) gene may be involved in bull spermatogenesis

BackgroundVasa is a member of the DEAD-box protein family that plays an indispensable role in mammalian spermatogenesis, particularly during meiosis. Bovine vasa homology (Bvh) of Bos taurus has been reported, however, its function in bovine testicular tissue remains obscure. This study aimed to reveal the functions of Bvh and to determine whether Bvh is a candidate gene in the regulation of spermatogenesis in bovine, and to illustrate whether its transcription is regulated by alternative splicing and DNA methylation.ResultsHere we report the molecular characterization, alternative splicing pattern, expression and promoter methylation status of Bvh. The full-length coding region of Bvh was 2190 bp, which encodes a 729 amino acid (aa) protein containing nine consensus regions of the DEAD box protein family. Bvh is expressed only in the ovary and testis of adult cattle. Two splice variants were identified and termed Bvh-V4 (2112 bp and 703 aa) and Bvh-V45 (2040 bp and 679 aa). In male cattle, full-length Bvh (Bvh-FL), Bvh-V4 and Bvh-V45 are exclusively expressed in the testes in the ratio of 2.2:1.6:1, respectively. Real-time PCR revealed significantly reduced mRNA expression of Bvh-FL, Bvh-V4 and Bvh-V45 in testes of cattle-yak hybrids, with meiotic arrest compared with cattle and yaks with normal spermatogenesis (P < 0.01). The promoter methylation level of Bvh in the testes of cattle-yak hybrids was significantly greater than in cattle and yaks (P < 0.01).ConclusionIn the present study, Bvh was isolated and characterized. These data suggest that Bvh functions in bovine spermatogenesis, and that transcription of the gene in testes were regulated by alternative splice and promoter methylation.

Effect of newborn bovine serum on cryopreservation of adult bovine testicular tissue

Bovine serum is widely used for cryopreservation of various cells and tissues. However, its cryoprotective effects on the cells and tissues are ambiguous and controversial. To test the effects of newborn calf serum (NCS) on cryopreservation of bovine testis tissue, NCS of 0%, 5%, 10% and 20% (v/v) was added into minimum essential medium + 10% dimethyl sulphoxide (DMSO)-based medium according to our previous report. Interestingly, the testicular cell viabilities and spermatogonia percentages from four groups were very close. The results indicated that an increase in the concentration of NCS in freezing medium to 20% has no significant effect on survival of both testicular cells and spermatogonia, and 10% DMSO-based freezing medium can maintain the testicular cell viability and spermatogonia percentage at a relatively high level (83.4 ± 0.7 and 56.5 ± 2.2 respectively). Taken together, NCS is dispensable for cryopreservation of adult bovine testis tissue. Our results provide an evidence for cutting down the costs in cryopreservation research of bovine testis tissue by reducing or giving up the use of serum.

Cell microarrays for the screening of factors that allow the enrichment of bovine testicular cells

Cell microarrays can serve as high-throughput platforms for the screening of a diverse range of biologically active factors and biomaterials that can induce desired cellular responses such as attachment, proliferation, or differentiation. Here, we demonstrate that surface-engineered microarrays can be used for the screening and identification of factors that allow the enrichment and isolation of rare cells from tissue-derived heterogeneous cell populations. In particular, we have focused on the enrichment of bovine testicular cells including type A spermatogonia and Sertoli cells. Microarray slides were coated with a copolymer synthesized from poly(ethylene glycol) methacrylate and glycidyl methacrylate to enable both the prevention of cell attachment between printed spots and the covalent anchoring of various factors such as antibodies, lectins, growth factors, extracellular matrix proteins, and synthetic macromolecules on printed spots. Microarrays were incubated with mixed cell populations from freshly isolated bovine testicular tissue. Overall, cell attachment was evaluated using CellTracker staining, whereas differential attachment of testicular cells was determined by immunohistochemistry staining with Plzf and vimentin antibodies as markers for type A spermatogonia and Sertoli cells, respectively. The results indicate that various surface immobilized factors, but in particular Dolichos biflorus lectin, allowed the enrichment of Plzf positive cells. Furthermore, Pisum sativum lectin, concanavalin A, collagen type IV, and vitronectin were identified as suitable negative selection factors. To our best knowledge, this work is the first to demonstrate the utility of surface engineered cell-based microarrays for the identification of factors that allow the selective capture of rare cells from tissue isolated heterogeneous mixtures.

Hyaluronidase for the management of dextrose extravasation

Intravenous catheter malfunction causing extravasation is a common occurrence in the emergency department (ED). A 17-year-old girl who had a syncopal episode was given an ampule of dextrose 50% water into her antecubital region by emergency medical services. During her evaluation at the ED, she developed erythema and swelling in the antecubital region where the dextrose was given and extravasation was suspected. Most extravasations have relatively minor sequelae, but once an extravasation has occurred, damage can continue for months and involve nerves, tendons, and joints. If treatment is delayed, surgical debridement, skin grafting, and even amputation have been reported in literature. Hyaluronidase is a parenteral protein enzyme traditionally derived from bovine testicular tissue. Hyaluronidase is used as adjunct therapy to increase the absorption and dispersion of other injected drugs. The drug has also been used as an adjunct to nonpharmacologic management of extravasation of selected antineoplastic agents and other irritants. Although hyaluronidase does not have a Food and Drug Administration indication for dextrose extravasation, literature has shown improved outcomes for other hyperosmolar substances. Hyaluronidase was used by injecting 0.2 mL (150 U/1 mL) with a 25-gauge needle at 5 different sites along the leading edge of erythema. The patient's symptoms improved, and there were no complications noted at follow-up at 48 hours and at 6 months. The use of hyaluronidase should be considered for the management of dextrose extravasations and other extravasations.

Immunohistochemical Localization of Relaxin‐Like Factor/Insulin‐Like Peptide‐3 in the Bovine Corpus Luteum

Relaxin-like factor/insulin-like peptide (INSL)-3 is highly expressed in the bovine corpus luteum throughout the estrous cycle and pregnancy. Demonstration of translation of the relaxin-like factor message was previously shown for the follicle but not the corpus luteum. In this study, relaxin-like factor mRNA was highly expressed in the corpus luteum on days 7 and 14 of pregnancy. Tissues were fixed in 10% neutral buffered formalin, and utilizing two different antibodies to relaxin-like factor, W3 rabbit anti-bovine and 2-8F mouse anti-bovine, relaxin-like factor was localized in fibroblast-like cells. Staining was also observed in the Leydig cells of bovine testicular tissue. No staining was observed in small and large steroidogenic luteal cells, indicating a nonsteroidogenic source of luteal relaxin-like factor. Definitive cell type identification is currently being determined via electron microscopy.

Pharmacology Review

Extravasation or inadvertent infiltration of fluids into subcutaneous tissue from peripheral intravenous (IV) devices is a common adverse event in newborns. (1)(2) Although fluids occasionally extravasate from central venous lines, the complication is much more common from peripheral catheters, which are used widely in sick neonates. (3) This article focuses on extravasations from peripheral IV devices. Injury to the skin, even in a very immature neonate, results in an inflammatory response and heals by scar formation. (4) Although the scars from many of these injuries appear to improve with time, (5) tissue necrosis from extravasation injury could result in partial or complete skin loss, infection, and nerve and tendon damage, with the potential risk of permanent cosmetic and functional impairment. (6)(7)(8) The incidence of extravasations from Teflon® catheters has been reported to vary from 23% to 63%. (1) A recent survey of regional neonatal intensive care units in the United Kingdom recorded the prevalence of extravasation injury resulting in skin necrosis as 38 per 1,000 neonates, with 70% of these injuries occurring in infants of 26 weeks’ gestation or less. (2) The incidence of extravasation is related closely to the type of device used, insertion site, duration of therapy, infusate administered, patient activity, and gestational age. (1)(3)(9)(10) The fragility of the skin, particularly in the first 2 weeks after birth, and the lack of subcutaneous tissue in preterm neonates makes them uniquely susceptible to injury and skin loss. Extravasation may occur from the tip of the cannula or needle piercing the vessel wall. Alternatively, distal obstruction of the vein due to thrombosis or venoconstriction from irritation of the vessel wall may lead to increased back pressure and leakage from the entry point of the needle or …

Establishment of spermatogenesis in neonatal bovine testicular tissue following ectopic xenografting varies with donor age.

Ectopic testicular xenografting can be used to investigate spermatogenesis and as an alternative means for generating transgenic spermatozoa in many species. Improving the efficiency of spermatogenesis in xenografted testicular tissue will aid in the application of using this approach. The present study was conducted to evaluate age-related differences in the establishment of spermatogenesis in grafted testicular tissue from bulls between 2 and 16 wk of life. Testicular tissue was ectopically xenografted under the skin on the backs of castrated nude mice and subsequently evaluated for growth, testosterone production, and establishment of spermatogenesis 24 wk after grafting. The greatest weight increases occurred in donor tissue from calves of the ages 2, 4, and 8 wk compared with the ages of 12 and 16 wk. Recipient mouse serum testosterone concentration was at normal physiological levels 24 wk after grafting and no significant differences were detected between recipients grafted with testicular tissue from bull calves of different ages. The development of germ cells to elongated spermatids were observed in seminiferous tubules of grafts from donor calves of the ages 4, 8, 12, and 16 wk but not observed in grafts from 2-wk donors, which contained round spermatids as the most advanced germ cell stage. Grafts from 8-wk donors contained a significantly higher (10-fold) average percentage of seminiferous tubules with elongated spermatids than all other donor ages. These data demonstrate differences in the ability of testicular tissue from donor animals of different ages to establish spermatogenesis following ectopic testicular xenografting.

Novel aspect of perinuclear theca assembly revealed by immunolocalization of non-nuclear somatic histones during bovine spermiogenesis.

The perinuclear theca (PT) is an important accessory structure of the sperm head, yet its biogenesis is not well defined. To understand the developmental origins of PT-derived somatic histones during spermiogenesis, we used affinity-purified antibodies against somatic-type histones H3, H2B, H2A, and H4 to probe bovine testicular tissue using three different immunolocalization techniques. While undetectable in elongating spermatid nuclei, immunoperoxidase light microscopy showed all four somatic histones remained associated to the caudal head region of spermatids from steps 11 to 14 of the 14 steps in bovine spermiogenesis. Immunogold electron microscopy confirmed the localization of somatic histones on two nonnuclear structures, namely transient manchette microtubules of step-9 to step-11 spermatids and the developing postacrosomal sheath of step-13 and -14 spermatids. Immunofluorescence demonstrated somatic histone immunoreactivity in the developing postacrosomal sheath, and on anti-beta-tubulin decorated manchette microtubules of step-12 spermatids. Focal antinuclear pore complex labeling on the base of round spermatid nuclei was detected by electron microscopy and immunofluorescence, occurring before the nucleoprotein transition period during spermatid elongation. This indicated that, if nuclear histone export precedes their degradation, this process could only occur in this region, thereby questioning the proposed role of the manchette in nucleocytoplasmic trafficking. Somatic histone immunodetection on the manchette during postacrosomal sheath formation supports a role for the manchette in PT assembly, signifying that some PT components have origins in the distal spermatid cytoplasm. Furthermore, these findings suggest that somatic histones are de novo synthesized in late spermiogenesis for PT assembly.

Spermatogenesis and germ cell transgene expression in xenografted bovine testicular tissue.

The present study was conducted to evaluate the development of spermatogenesis and utility of using electroporation to stably transfect germ cells with the beta-galactosidase gene in neonatal bovine testicular tissue ectopically xenografted onto the backs of recipient nude mice. Bull testicular tissue from 4-wk donor calves, which contains a germ cell population consisting solely of gonocytes or undifferentiated spermatogonia, was grafted onto the backs of castrated adult recipient nude mice. Testicular grafts significantly increased in weight throughout the grafting period and the timing of germ cell differentiation in grafted tissue was consistent with postnatal testis development in vivo relative to the bull. Seminiferous tubule diameter also significantly increased with advancing time after grafting. At 1 wk after grafting, gonocytes in the seminiferous cords completed migration to the basement membrane and differentiated germ cell types could be observed 24 wk after grafting. The presence of elongating spermatids at 24 wk confirmed that germ cell differentiation occurred in the bovine tissue. Leydig cells in the grafted bovine tissue were also capable of producing testosterone in the castrated recipient mice from 4 wk to 24 wk after grafting at concentrations that were similar to levels in intact, nongrafted control mice. The testicular tissue that had been electroporated with a beta-galactosidase expression vector showed tubule-specific transgene expression 24 wk after grafting. Histological analysis showed that transgene expression was present in both Sertoli and differentiated germ cells but not in interstitial cells. The system reported here has the potential to be used for generation of transgenic bovine spermatozoa.

The PrP-like protein Doppel gene in sheep and cattle: cDNA sequence and expression.

cDNAs encoding the ovine and bovine prion protein-like protein Doppel (Dpl) have been cloned. Sequencing revealed cDNAs of 2.85 and 3.31 kb from ovine and bovine testicular tissue, in accordance with observations of single transcripts of 3.2 and 3.6 kb on Northern blots. Sequence alignments showed a very high degree of identity between the sheep and cattle Dpl cDNAs, except for a 0.4-kb stretch in the bovine 3' untranslated region and the terminal 3' end of the sequences. The expression pattern of the Dpl gene (Prnd) in adult tissues from both species was compared by Northern blot and RT-PCR analyses. The Prnd gene was expressed strongly in testicular tissue, while low levels of expression were seen in other tissues. The open reading frame of the ovine and bovine sequences encodes a 178-amino acid protein with 95% sequence identity between the two species. Predicted structural features are in close agreement with previous reports for mouse, human, and rat Dpl.

Distribution Pattern of F-Actin, Vimentin and Alpha-Tubulin in the Bovine Testis during Postnatal Development

The distribution of F-actin, vimentin and alpha-tubulin was studied immunohistochemically in bovine seminiferous and straight testicular tubules, rete testis and intertubular tissue during postnatal development. Sites of antigenicity were detected by ABC immunoperoxidase technique and visualized by metal-enhanced deposition of diaminobenzidine. Within the seminiferous epithelium, F-actin appears at 20 weeks and is found in adult Sertoli cells as part of specialized cell contacts. In peritubular cells, F-actin increases gradually from 4 to 30 weeks when the adult concentration is achieved. After 20 weeks, subepithelial fibroblasts of the mediastinum testis start to express F-actin and at 52 weeks, a thick layer of positive myofibroblasts is seen beneath the epithelia of rete testis and straight testicular tubules. Testicular macrophages and light intercalated cells (LIC) are also characteristically decorated following F-actin immunoreaction. Vimentin is localized in perinuclear position in pre-Sertoli cells of 4-20 weeks and in adult Sertoli cells. During the period of transformation from pre-Sertoli to Sertoli cells, the perinuclear vimentin coat is absent. The epithelia of rete testis and straight tubules exhibit a strong vimentin immunoreaction in their basal parts. This specific pattern does not change from 4 weeks to adulthood. Alpha -tubulin is absent in 4-week-old seminiferous tubules. At 8 weeks, the perinuclear area of pre-Sertoli cells reacts positive. The alpha-tubulin content increases in these cells continuously, and from 30 weeks on nearly the entire supranuclear cytoplasm of Sertoli cells is heavily decorated. The epithelial of rete and straight tubules display a growing number of alpha-tubulin-positive cells from 4 to 40 weeks. From then on, nearly all epithelial cells contain alpha-tubulin, particularly in a narrow zone beneath their lateral cell borders.

Testicular Oxytocin Gene Expression in Seminiferous Tubules of Cattle and Transgenic Mice*

We are using transgenic mice to study the regulation of the bovine vasopressin (VP) and oxytocin (OT) genes. Prompted by the observation that mice bearing a bovine OT transgene express bovine OT RNA in their testes, we investigated the expression of the VP-OT locus in normal mice and cattle. Normal wild-type mice do not have detectable levels of either VP or OT RNA in their testes. Normal cattle are also devoid of detectable VP transcripts, but have relatively high levels of testicular OT RNA. Additionally, OT, but not VP, peptide is detectable by HPLC. In situ hybridization to RNA in bovine testicular tissue sections localized OT transcripts to seminiferous tubules, with a distribution similar to that of alpha-inhibin, suggesting expression in Sertoli cells. Interestingly, the bovine OT RNAs in the transgenic mouse testes were also shown by in situ hybridization to have the same distribution. These data suggest that the cis-acting regulatory sequences responsible for expression of the OT gene in bovine Sertoli testis reside within the limits of the transgene used in this study. Further, the trans-acting factors present in murine testicular cells are able to recognize these elements, although they do not express the endogenous mouse OT gene in this tissue.

Gas chromatographic and gas chromatographic/tandem mass spectrometric characterization of precursors on the sesterterpene pathway for steroid biosynthesis.

An increasing amount of evidence is accumulating to support the proposal that steroidogenesis can occur by a sesterterpene pathway as well as the cholesterol pathway. Key intermediates on the sesterterpene pathway are 23,24-dinor-5-cholen-3 beta-ol (guneribol) and some of its metabolites, e.g. 23,24-dinor-4-cholen-3-one (guneribone). It has been reported that these intermediates are biosynthesized and converted to steroid hormones by a range of endocrine tissues in vitro. Monitoring the pentafluorobenzyloxime derivatives by negative ion chemical ionization mass spectrometry in the electron capture mode provided evidence for the presence of guneribone in extracts of bovine testicular and human adrenal tumour tissue. Complementary evidence was obtained from gas chromatographic/tandem mass spectrometric data generated on a triple-quadrupole instrument by monitoring daughter ions (in the multiple ion detection, MID mode) of the molecular anion of derivatized guneribone in both standards and tissue extracts. The present findings that sesterterpene pathway intermediates are present as endogenous compounds in tissue extracts, together with the previously reported radiochemical data, give further support to the sesterterpene pathway hypothesis.

Characterization of adenosine binding sites in bovine testicular tissue using 8-cyclopentyl-1,3-[ 3H]dipropylxanthine

The existence of specific adenosine binding sites in bovine testicular tissue was evaluated using the novel antagonist radioligand 8-cyclopentyl-1,3-[ 3H]dipropylxanthine ([ 3H]DPCPX). Saturation analysis revealed specific binding that was saturable at ∼ 1 nM. Scatchard analysis indicated a single class of binding sites with K D = 0.26 nM and a B max = 0.37 pmol/mg protein. Affinity profiles suggest an A 1 subtype recognition site that is different from the classical A 1 adenosine receptor. The results presented should prove useful in subsequent studies concerning heterogeneity among adenosine receptors and also aid in discerning the role of adenosine in reproduction.