Buffalo Testis Research Articles

Analysis of OCT4 and PGP9.5 gene expression in prenatal and postnatal buffalo (Bubalus bubalis) testes

This study aimed to investigate and characterize the spermatogonial stem cells (SSCs) in buffaloes at different stages of development, including prenatal, neonatal, prepubertal, and adult testes. We sought a comprehensive understanding of these cells through a combination of histological, immunohistochemical, and ultrastructural analyses. Specifically, we examined changes in the expression of two potential SSC markers, OCT4 and PGP9.5, using immunohistochemistry. Additionally, we conducted a real-time quantitative polymerase chain reaction (RT-qPCR) to assess the relative gene expression of OCT4 and PGP9.5. The relative expression of the OCT4 gene was down-regulated in the adult testes compared to its expression during prepubertal and neonatal life. The relative expression of the PGP9.5 gene was up-regulated in the neonatal testes and down-regulated in the prepubertal and adult testes. The spermatogonia were round, oval-to-ellipsoidal cells lying over the basement membrane (BM) with a round-to-oval nucleus. Based on the immunoexpression of the putative SSC markers, OCT4 and PGP9.5, we concluded that the proportion of stem cells was highest during the neonatal stage, followed by the prepubertal and prenatal stages. This finding sheds light on the dynamics of spermatogonial stem cells in buffalo testes at different developmental stages, providing valuable insights into these cells’ regulation and potential applications.

Single-cell RNA sequencing uncovers dynamic roadmap and cell-cell communication during buffalo spermatogenesis

Spermatogenesis carries the task of precise intergenerational transmission of genetic information from the paternal genome and involves complex developmental processes regulated by the testicular microenvironment. Studies performed mainly in mouse models have established the theoretical basis for spermatogenesis, yet the wide interspecies differences preclude direct translation of the findings, and farm animal studies are progressing slowly. More than 32,000 cells from prepubertal (3-month-old) and pubertal (24-month-old) buffalo testes were analyzed by using single-cell RNA sequencing (scRNA-seq), and dynamic gene expression roadmaps of germ and somatic cell development were generated. In addition to identifying the dynamic processes of sequential cell fate transitions, the global cell-cell communication essential to maintain regular spermatogenesis in the buffalo testicular microenvironment was uncovered. The findings provide the theoretical basis for establishing buffalo germline stem cells invitro or culturing organoids and facilitating the expansion of superior livestock breeding.

Improved quality and fertilizability of cryopreserved buffalo spermatozoa with the supplementation of methionine sulfoxide reductase A.

The excessive reactive oxygen species produced during semen-freezing and -thawing damage the macromolecules resulting in impairment of cellular functions. Proteins are the primary targets of oxidative damage, wherein methionine residues are more prone to oxidation and get converted into methionine sulfoxide, thus affecting the protein function. The methionine sulfoxide reductase A (MsrA) catalyzes the conversion of methionine sulfoxide to methionine and restores the functionality of defective proteins. To establish the expression of MsrA in male reproductive organs, including semen and its effect on quality of cryopreserved semen upon exogenous supplementation, taking buffalo semen as a model. The expression of MsrA was established by immunohistochemistry, PCR, and Western blots. Further, the effect of recombinant MsrA (rMsrA) supplementation on the quality of cryopreserved spermatozoa was assessed in three treatment groups containing 1.0, 1.5, and 2.0µg of rMsrA/50 million spermatozoa in egg yolk glycerol extender along with a control group; wherein the post-thaw progressive motility, viability, membrane integrity, and zona binding ability of cryopreserved spermatozoa were studied. The MsrA was expressed in buffalo testis, epididymis, accessory sex glands, and spermatozoa except in seminal plasma. In group 2, the supplementation has resulted in a significant (p<0.05) improvement as compared to the control group in mean progressive motility (47.50 ± 2.50 vs. 36.25 ± 2.63), viability (56.47 ± 1.85 vs. 48.05 ± 2.42), HOST (50.76 ± 1.73 vs. 44.29 ± 1.29), and zona binding ability of spermatozoa (149.50 ± 8.39 vs. 29.50 ± 2.85). In the absence of native MsrA of seminal plasma, the supplementations of rMsrA may repair the oxidatively damaged seminal plasma proteins and exposed sperm plasma membrane proteins resulting in better quality with a fivefold increase in fertilizability of frozen-thawed spermatozoa. The findings can be extended to other species to improve the semen quality with the variation in the amounts of rMsrA supplementation.

Isolation, Cultivation and Identification of Spermatogonial Stem Cells from Juvenile Buffalo Testes

Isolation, Cultivation and Identification of Spermatogonial Stem Cells from Juvenile Buffalo Testes

Novel targets identified by integrated proteomic and phosphoproteomic analysis in spermatogenesis of swamp buffalo (Bubalus bubalis)

To understand mechanisms of spermatogenesis, the proteome and the phosphoproteome in prepubertal and pubertal swamp buffalo (Bubalus bubalis) testes were analyzed using tandem mass tag (TMT) coupled with liquid chromatography-tandem mass spectrometry (LC–MS/MS). In prepubertal testes, 80 proteins were overexpressed, 148 proteins were underexpressed, and 139 and 142 protein sites had higher and lower phosphorylation, respectively, compared to the levels in pubertal testes. Several of these proteins were associated with reproductive processes such as sexual reproduction, spermatogenesis, fertilization, and spermatid development. In particular, outer dense fiber protein 1 (ODF1), protein maelstrom homolog (MAEL), actin-like protein 7B (ACTL7B), tyrosine-(Y)-phosphorylation regulated (CABYR), and tripartite motif containing 36 (TRIM36) were upregulated with age at both the proteome and phosphoproteome levels. Combining proteome and phosphoproteome analysis can be effectively applied to study the protein/phosphorylation patterns of buffalo testes. These data provide new regulatory candidates and evidence for a complex network in spermatogenesis in buffalo testes, and serve as an important resource for exploring the physiological mechanism of spermatogenesis in mammals.

Homologous transplantation of fluorescently labelled enriched buffalo (Bubalus bubalis) spermatogonial stem cells to prepubertal recipients

Spermatogonial stem cell transplantation provides a unique opportunity to study the biology of spermatogenesis and also offers an alternative approach for genetic modification in large animals. The present study aimed to extend this technique to the water buffalo. Spermatogonial stem cells (SSCs) were isolated from prepubertal buffalo testes (3-6 months of age) using two-step enzymatic digestion method and enriched by differential plating and Percoll density gradient centrifugation. The enriched SSCs expressed numerous spermatogonial transcriptional markers, viz. ID4, THY1, BCL6B, UCHL1, ETV5 and REX1 which confirmed their bonafide SSC identity. Subsequently, the enriched SSCs were labelled with a fluorescent dye PKH26 and transplanted into buffalo calves under ultrasound guidance. The recipient testes were recovered after 7-8 weeks by castration and their fluorescence microscopebased examination exhibited the persistence and localization of the fluorescent donor cells within the recipient seminiferous tubules. Further validation was done by the flow cytometric evaluation of PKH26 labeled donor cells among those isolated by two-step enzymatic digestion of recipient testicular parenchyma. In conclusion, we demonstrated the feasibility of SSC transplantation technique in the water buffalo.

Ubiquitome analysis reveals the involvement of lysine ubiquitination in the spermatogenesis process of adult buffalo (Bubalus bubalis) testis.

Protein ubiquitination, a major and conserved post-translational modification, is known to play a critical regulatory role in many biological processes in eukaryotes. Although several ubiquitinated proteins have been found in buffalo (Bubalus bubalis) testis in our previous studies, large-scale profiling of buffalo testis ubiquitome has not been reported to date. In the present study, we first identified a global profiling of lysine ubiquitination of adult buffalo testis using a highly sensitive LC-MS/MS coupled with immune-affinity enrichment of ubiquitinated peptides. In total, 422 lysine ubiquitination sites were identified in 262 proteins in adult buffalo testis tissue. Bioinformatics analysis showed that the ubiquitinated proteins are involved in a variety of biological processes and diverse subcellular localizations. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and protein interaction network analysis indicated that proteasome, glycolysis/gluconeogenesis and gap junction pathways are modulated by protein ubiquitination in testis. Besides, 44 ubiquitinated proteins may involve in spermatogenesis according to the SpermatogenesisOnline database, of which, the ubiquitination of HSPA2 and UCHL1 were confirmed by Immunoprecipitation (IP)/Western blot analysis. Taken together, these data provide a global view of ubiquitome in buffalo testis for the first time, and serve as an important resource for exploring the physiological role especially spermatogenesis of lysine ubiquitination in testis in mammals.

198 SUCCESSFUL TRANSPLANTATION OF BUFFALO (Bubalus bubalis) GERM CELLS TO HOMOLOGOUS RECIPIENTS

Transplantation of isolated germ cells from a fertile donor male into the seminiferous tubules of infertile recipients can result in donor germ cells-derived sperm production. This technique has the potential to be used as an alternative strategy for producing transgenic livestock with higher efficiency and less time and capital requirement than the current methods. The objective of the present study was to investigate whether the homologous transplantation of isolated buffalo germ cells could result in colonization of recipient testes. Germ cells were isolated from prepubertal buffalo testes (4–6 months of age) by using double enzymatic digestion method and filtration through 80- and 60-µm nylon mesh filters. Further enrichment was achieved by differential plating on Datura stramonium agglutinin lectin-coated dishes and after that Percoll density gradient centrifugation as descrived by van Pelt et al. (1996) with minor modifications. A discontinuous density gradient was prepared with 60, 50, 40, 36, 34, 32, 30, 28, and 20% Percoll in a 15-mL centrifuge tube. The enriched germ cells were then labelled with red fluorescent linker dye PKH26 (Sigma, St. Louis, MO, USA) as per the manufacturer’s instructions, and ~10 million cells/testis were transferred into the rete testis of 3 recipients (16–18 months of age) under ultrasonographic guidance. After 45 days, testes were surgically removed and samples were prepared for analysis of labelled cells via wet mount of seminiferous tubules and individual cells isolation. When wet mount specimen were observed under a fluorescence microscope, PKH26-positive cells were identified on the seminiferous tubule basement membrane in all 3 recipients, which indicated that these cells had successfully migrated from the tubule lumen and were likely to be donor germ cells. In freshly isolated cells, clumps of PKH26-positive cells were observed, which indicated either cell division or extensive local colonization of specific areas of the seminiferous tubules. In conclusion, we report successful homologous transplantation of germ cells in prepubertal buffalo testes. Further studies will investigate functionality of transferred testicular cells.

Replacement of serum with ocular fluid for cryopreservation of immature testes

Cryopreservation of immature testis is a feasible approach for germplasm preservation of male animals. Combinations of dimethyl sulfoxide (DMSO) and foetal bovine serum (FBS) are used for testis cryopreservation. However, an alternative to FBS is needed, because FBS is expensive. Buffalo ocular fluid (BuOF), a slaughter house by-product, could be an economical option. The objective of the present study was to assess whether BuOF can replace FBS for cryopreservation of immature mouse (Mus musculus), rat (Rattus norvegicus), and buffalo (Bubalus bubalis) testes. Results showed that rodent and buffalo testes frozen in DMSO (10% for rodents and 20% for buffalo) with 20% FBS or BuOF had similar numbers of viable and DNA-damaged cells (P > 0.05). The expression of cell proliferation- (PCNA) and apoptosis-specific proteins (Annexin V and BAX/BCL2 ratio) were also comparable in mouse and buffalo testes frozen in DMSO with FBS or BuOF (P > 0.05). Interestingly, rat testis frozen in DMSO with BuOF had lower expression of Annexin V protein than testis frozen in DMSO with FBS (P < 0.05). The percentage of meiotic germ cells (pachytene-stage spermatocytes) in xenografts from testis frozen either in DMSO with BuOF or FBS did not significantly differ in rats or buffalo (P > 0.05). These findings provide evidence that BuOF has potential to replace FBS for cryopreservation of immature rodent and buffalo testis. Further investigation is needed to explore whether BuOF can replace FBS for testis cryopreservation of other species.

Purification of Regucalcin from the Seminal Vesicular Fluid: A Calcium Binding Multi-Functional Protein.

Regucalcin is a multi-functional protein having roles in calcium homeostasis as well as in anti-apoptotic, anti-prolific and anti-oxidative functions. Recently, it has been reported from the male reproductive tract, but its role in male reproduction needs further investigation; for which the native regucalcin of reproductive origin will be more appropriate. The gel exclusion chromatography followed by diethyl aminoethane cellulose chromatography and two-dimentional cellulose acetate membrane electrophoresis used for its purification are time consuming and less specific. Here, the regucalcin gene from buffalo testis has been cloned, expressed and purified in recombinant form, and subsequently used for raising hyper-immune serum. The Western blot of seminal vesicular fluid probed with anti-regucalcin polyclonal and monoclonal antibodies showed the presence of 28 and 34kDa bands specific to regucalcin. Further, an affinity matrix has been prepared using anti-regucalcin polyclonal antibodies. An immuno-affinity chromatography method has been standardized to isolate regucalcin from seminal vesicular fluid. The initial complexity of the protein mixture in the seminal vesicular fluid has been reduced by a heat coagulation step. The purified protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band at 68kDa that has been further confirmed as regucalcin by Liquid chromatography-mass spectrometry/mass spectrometry. The RGN purified from seminal vesicular fluid will be more appropriate for studying its possible role in male reproduction, especially sperm cell capacitation, hyperactivation, acrosome reaction and cryopreservation. The study can be applied in purifying regucalcin from different tissues or species with minor modifications in the methodology.

Isolation and Identification of Prepubertal Buffalo (Bubalus bubalis) Spermatogonial Stem Cells.

Isolation and culture of spermatogonial stem cells (SSCs) are attractive for production of genetic modified offspring. In the present study, buffalo spermatogonial stem-like cells were isolated, cultured and expression pattern of different germ cell marker genes were determined. To recover spermatogonia, testes from age 3 to 7 months of buffalo were decapsulated, and seminiferous tubules were enzymatically dissociated. Two types of cells, immature sertoli cell and type A spermatogonia were observed in buffalo testes in this stage. Germ cell marker genes, OCT3/4 (Pou5f1), THY-1, c-kit, PGP9.5 (UCHL-1) and Dolichos biflorus agglutinin, were determined to be expressed both in mRNA and protein level by reverse transcription polymerase chain reaction and immunostaining in buffalo testes and buffalo spermatogonial stem-like cells, respectively. In the following, when the isolated buffalo buffalo spermatogonial stem-like cells were cultured in the medium supplemented 2.5% fetal bovine serum and 40 ng/mL glial cell-derived neurotrophic factor medium, SSCs proliferation efficiency and colony number were significantly improved than those of other groups (p<0.05). These findings may help in isolation and establishing long term in vitro culture system for buffalo spermatogonial stem-like cells, and accelerating the generation of genetic modified buffaloes.

Molecular characterization and expression of buffalo (Bubalus bubalis) DEAD-box family VASA gene and mRNA transcript variants isolated from testis tissue

VASA is a member of the DEAD-box protein family that plays an indispensable role in mammalian spermatogenesis, particularly during meiosis. In the present study, we isolated, sequenced, and characterized VASA gene in buffalo testis. Here, we demonstrated that VASA mRNA is expressed as multiple isoforms and uses four alternative transcriptional start sites (TSSs) and four different polyadenylation sites. The TSSs identified by 5′-RNA ligase-mediated rapid amplification of cDNA ends (RLM-5′-RACE) were positioned at 48, 53, 85, and 88 nucleotides upstream relative to the translation initiation codon. 3′-RACE experiment revealed the presence of tandem polyadenylation signals, which lead to the expression of at least four different 3′-untranslated regions (209, 233, 239 and 605 nucleotides). The full-length coding region of VASA was 2190bp, which encodes a 729 amino acid (aa) protein containing nine consensus regions of the DEAD box protein family. VASA variants are highly expressed in testis of adult buffalo. We found five variants, one full length VASA (729 aa) and four splice variants VASA 2, 4, 5, 6 (683, 685, 679, 703 aa). The expression level of VASA 1 was significantly higher than rest of all (P<0.05) except VASA 6. The relative ratio for VASA 1:2:4:5:6 was 100:1.0:1.6:0.9:48.

Identification and IVC of spermatogonial stem cells in prepubertal buffaloes

Development of suitable selective marker for buffalo spermatogonial stem cells (SSCs), optimization of long-term IVC conditions, and their pluripotent retention capacity in buffaloes can be of prime importance in selective genetic modifications of this species. In the present study, we identified CDH1 as a specific marker for buffalo SSCs and revealed that it existed in two protein isoforms (large [135 kDa] and small [90 kDa] subunits) in the buffalo testis; furthermore, immunohistochemical analysis revealed that CDH1 expression was present in spermatogonia but absent in the somatic cells of 4-month-old buffalo testis. After 7 days of enrichment, expression of CDH1 was also detectable in IVC colonies (∼53% enrichment efficiency by Fluorescence-activated cell sorting (FACS)). For long-term culture of SSCs, proliferation studies with different factors showed that combination of 20 ng/mL GDNF, 10 ng/mL FGF2, and 1000 U/mL LIF could significantly promote number of colonies (∼two folds) and proliferation of buffalo SSCs (∼three folds) compared with those of control or single-treatment groups; furthermore, addition of these combination growth factors significantly upregulated the messenger RNA level of spermatogonial-specific and pluripotency-related markers (BCL6B, GFRA1, and POU5F1), whereas downregulated receptor tyrosine kinase (KIT). For confirmation of their stem cell potential, Dolichos biflorus agglutinin–stained cells were identified in the basal membrane of seminiferous tubules of xenotransplanted mice testis. These findings indicate the identification of a new buffalo SSCs marker; furthermore, it may help in establishing long-term culture that would assist in genetic modification of these buffaloes.

Prepubertal Buffalo (Bubalus bubalis) Leydig Cells: Isolation, Culture and Characterization

Water buffalo (Bubalus bubalis) is an economically important livestock species in India. Male buffaloes display delayed sexual maturity as compared to the bulls (Bos taurus). Serum testosterone level, the key regulator of sexual maturity of males, is reported to be low in male buffaloes in comparison to bulls. Testosterone secretion and progression of spermatogenesis is mediated essentially by Leydig cells in the males. Establishment of primary culture for buffalo Leydig cells can provide an excellent tool to investigate the factors which regulate testicular steroidogenesis. Therefore, the objectives of the present study were to isolate, culture and characterize buffalo Leydig cells. Immunohistological analysis revealed that cytochrome P450, family 11, subfamily A, polypeptide 1 (CYP11A1) specifically mark the Leydig cells in prepubertal buffalo testis. Using enzymatic digestion and Percoll density gradient centrifugation, a cell population that consisted of approximately 95% pure Leydig cells was obtained as indicated by CYP11A1 staining. Purified Leydig cells were cultured in DMEM/F12 supplemented with 10% foetal bovine serum (FBS) for 72 h. The cultured Leydig cells proliferated, expressed Leydig-cell specific transcripts (STAR, HSD3B1, HSD3B6, and CYP17A1) and proteins (CYP11A1, HSD3B and LHCGR), and secreted testosterone. It was concluded from the present study that buffalo Leydig cells can be maintained in culture for 72 h. The primary culture of buffalo Leydig cells can be used for studying acute responses, biochemical properties and other factors regulating testicular steroidogenesis, independent of other testicular cell types.

Effects of glial cell line-derived neurotrophic factor, fibroblast growth factor 2 and epidermal growth factor on proliferation and the expression of some genes in buffalo (Bubalus bubalis) spermatogonial cells

The present study evaluated the effects of glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor (FGF) 2 and epidermal growth factor (EGF) on proliferation and the expression of some genes in spermatogonial cells. Spermatogonial cells were isolated from prepubertal buffalo testes and enriched by double enzyme treatment, filtration through 80- and 60-μm nylon mesh filters, differential plating on lectin-coated dishes and Percoll density gradient centrifugation. Cells were then cultured on a buffalo Sertoli cell feeder layer and formed colonies within 15-18 days. The colonies were found to predominantly contain undifferentiated Type A spermatogonia because they bound Dolichos biflorus agglutinin and did not express c-kit. The colonies expressed alkaline phosphatase, NANOG, octamer-binding transcription factor (OCT)-4 and tumour rejection antigen (TRA)-1-60. Cells were subcultured for 15 days, with or without growth factor supplementation. After 15 days, colony area and the relative mRNA abundance of PLZF were higher (P<0.05) following supplementation with 40 ng mL⁻¹ GDNF + 10 ng mL⁻¹ EGF + 10 ng mL⁻¹ FGF2 than with the same concentrations of GDNF alone or GDNF plus either EGF or FGF2. Expression of TAF4B was higher (P<0.05) in the presence of FGF2, whereas the expression of THY1 was not affected by growth factor supplementation. In the Sertoli cell feeder layer, EGF and FGF2 decreased (P<0.05), whereas GDNF increased (P<0.05), the relative mRNA abundance of ETV5 compared with control. In conclusion, an in vitro culture system that incorporates various growth factors was developed for the short-term culture of buffalo spermatogonia.

Isolation and enrichment of type A spermatogonia from pre-pubertal buffalo (Bubalus bubalis) testis

The aim of this study was to isolate and subsequently enrich type A spermatogonia from pre-pubertal buffalo testis. Two-step enzymatic digestion was used to isolate spermatogonia from 10 to 14months pre-pubertal buffalo calves, resulting in maximal release of spermatogonia from the seminiferous tubules. After enzymatic digestion, the type A spermatogonia were subsequently enriched by differential plating and Percoll gradient centrifugation. The identity of type A spermatogonia was determined by light microscopy and further characterised by Dolichos biflorus agglutinin, a specific marker for bovine type A spermatogonia by fluorescence-activated cell sorting analysis. After enzymatic isolation, the cell suspension contained about 27% of type A spermatogonia, which was enriched up to 71% with >70% cell viability. Further flow cytometric analysis showed the presence of THY1+ cells (cells expressing thymocyte differentiation antigen 1), suggesting that THY1 is a conserved marker of the undifferentiated spermatogonial cells in buffalo. The isolation of the enriched type A spermatogonia from buffalo testis opens ways to study the further biochemical characteristics of this important class of germ cells in this species.

Spermatogonial Stem Cells (SSCs) in Buffalo (Bubalus bubalis) Testis

BackgroundWater buffalo is an economically important livestock species and about half of its total world population exists in India. Development of stem cell technology in buffalo can find application in targeted genetic modification of this species. Testis has emerged as a source of pluripotent stem cells in mice and human; however, not much information is available in buffalo.Objectives and MethodsPou5f1 (Oct 3/4) is a transcription factor expressed by pluripotent stem cells. Therefore, in the present study, expression of POU5F1 transcript and protein was examined in testes of both young and adult buffaloes by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical analysis. Further, using the testis transplantation assay, a functional assay for spermatogonial stem cells (SSCs), stem cell potential of gonocytes/spermatogonia isolated from prepubertal buffalo testis was also determined.ResultsExpression of POU5F1transcript and protein was detected in prepubertal and adult buffalo testes. Western blot analysis revealed that the POU5F1 protein in the buffalo testis exists in two isoforms; large (∼47 kDa) and small (∼21 kDa). Immunohistochemical analysis revealed that POU5F1 expression in prepubertal buffalo testis was present in gonocytes/spermatogonia and absent from somatic cells. In the adult testis, POU5F1 expression was present primarily in post-meiotic germ cells such as round spermatids, weakly in spermatogonia and spermatocytes, and absent from elongated spermatids. POU5F1 protein expression was seen both in cytoplasm and nuclei of the stained germ cells. Stem cell potential of prepubertal buffalo gonocytes/spermatogonia was confirmed by the presence of colonized DBA-stained cells in the basal membrane of seminiferous tubules of xenotransplanted mice testis.Conclusion/SignificanceThese findings strongly indicate that gonocytes/spermatogonia, isolated for prepubertal buffalo testis can be a potential target for establishing a germ stem cell line that would enable genetic modification of buffaloes.

Gonadal status of male recipient mice influences germ cell development in immature buffalo testis tissue xenograft

Growth and development of immature testis xenograft from various domestic mammals has been shown in mouse recipients; however, buffalo testis xenografts have not been reported to date. In this study, small fragments of testis tissue from 8-week-old buffalo calves were implanted subcutaneously onto the back of immunodeficient male mouse recipients, which were either castrated or left intact (non-castrated). The xenografts were retrieved and analyzed 12 and 24 weeks later. The grafted tissue survived and grew in both types of recipient with a significant increase in weight and seminiferous tubule diameter. Recovery of grafts from intact recipients 24 weeks post-grafting was significantly lower than that from the castrated recipients. Seminal vesicle indices and serum testosterone levels were lower in castrated recipients at both collection time points in comparison to the intact recipients and non-grafted intact mouse controls. Pachytene spermatocytes were the most advanced germ cells observed in grafts recovered from castrated recipients 24 weeks post-grafting. Complete spermatogenesis, as indicated by the presence of elongated spermatids, was present only in grafts from intact recipients collected 24 weeks post-grafting. However, significant number of germ cells with DNA damage was also detected in these grafts as indicated by TUNEL assay. The complete germ cell differentiation in xenografts from intact recipients may be attributed to efficient Sertoli cell maturation. These results suggest that germ cell differentiation in buffalo testis xenograft can be completed by altering the recipient gonadal status.

Spermatogonia-specific proteins expressed in prepubertal buffalo ( Bubalus bubalis) testis and their utilization for isolation and in vitro cultivation of spermatogonia

Buffalo is an economically important livestock species in Asia. Little is known about male germ line technology owing to lack of sufficient understanding regarding expression of germ- and somatic-cell specific-proteins in the testis. In this study, we identified UCHL-1 (PGP 9.5) and lectin- Dolichos biflorus agglutinin (DBA) as specific markers for spermatogonia in buffalo testis. Expression of germ-cell and pluripotency-specific proteins such as DDX4 (VASA) and POU5F1 (OCT3/4) were also present in spermatogonia. Interestingly, the expression of somatic cell-specific proteins such as VIMENTIN and GATA4 were also detected in germ cells. Using two-step enzymatic digestion followed by differential plating and Percoll density-gradient centrifugation, an approximately 55% spermatogonia-enriched cell population could be obtained from the prepubertal buffalo testis. Isolated spermatogonia could survive and proliferate in vitro in DMEM/F12 medium containing 10% fetal bovine serum in the absence of any specific growth factors for a week. Cultured spermatogonia showed DBA affinity and expressed DDX4 and POU5F1. These results may help to establish a long-term culture system for buffalo spermatogonia.

The autonomous innervation of the buffalo (Bubalus bubalis) testis. An immunohistochemical study.

The innervation pattern in the buffalo testis was determined by using histochemical and immunohistochemical methods. Nerves were concentrated in the tunica albuginea and septula testis, and did not show an uniform distribution. The tunica albuginea at the lateral and medial sides and at the free border of the testis is most densely innervated than at the epididymal border. At the cranial pole thick nerve bundles were observed between albugineal vessels and muscle bundles. Rare parenchymal nerves were found in perivascular position between seminiferous tubules and their occurrence is confined to lobules at the cranial and caudal testicular poles. An intense NPY immunoreactivity occurred in nerve bundles and in solitary varicose fibres. Nerves were concentrated in the tunica albuginea at the lateral and medial side and at the free border of the testis, and in the lobules at the cranial and caudal testicular poles. Sub P immunoreactivity was occasionally detected in some thicker nerve bundles and solitary fibers, in the tunica albuginea and in the wall of blood vessels, showing a similar distribution but less intensity and density than NPY immunoreactivity. TH immunoreactivity stained nerve fibers in the buffalo testis with a distribution pattern similar to that obtained with general neuronal markers. The histochemical reaction for AchE was negative, so cholinergic fibers cannot be detected in the buffalo testis. The histochemical NADPHd reaction stained rare nitrergic nerve bundles and solitary fibers. The majority of NADPHd activity was confined to the vascular endothelium, and rarely to the interstitial Leydig cells, whereas the Sertoli and germ cells did not show any reaction.