Kisspeptin in the early post-partum of the dairy cow.

Recently, a new molecule, kisspeptin (Kp), and in particular Kisspeptin 10 (Kp10), was implicated in stimulating the hypothalamic-pituitary-gonadal axis. The aim of this study was to evaluate circulating Kp10 levels in the early post-partum period of the dairy cow. Blood samples were collected from 40 dairy cows, at 10 (T10), 12 (T12), 14 (T14) and 16 (T16) days after calving. Progesterone (P4) levels were evaluated using ELISA, and levels of oestrogens (E2) and Kp were evaluated using a radio-immunologic method. After an initial plateau, Kp10 significantly increased at T14 and decreased at T16. The P4 and E2 mean serum values remained in the physiological range. It is likely that Kp10 enhanced hypothalamic GnRH release as well as pituitary gonadotropin secretion, thus promoting follicular growth and the increase in E2 levels, which might have further enhanced Kp10 release through a positive feedback loop. To our best knowledge, this is the first report on the range of Kp10 blood concentration during the early post-partum period in the dairy cow. The results of our study will increase our current understanding of the complex neuro-endocrine crosstalk underlying the resumption of ovarian cyclicity in the dairy cow.

Effect of cariporide on ram sperm pH regulation and motility: possible role of NHE1.

Sperm motility, a feature essential for in vivo fertilization, is influenced by intracellular pH (pHi) homeostasis. Several mechanisms are involved in pHi regulation, among which sodium-hydrogen exchangers (NHEs), a family of integral transmembrane proteins that catalyze the exchange of Na+ for H+ across lipid bilayers. A preliminary characterization of NHE activity and kinetic parameters, followed by analysis of the expression and localization of the protein in ram spermatozoa was performed. NHE activity showed an apparent Km for external Na+ of 17.61 mM. Immunoblotting revealed a molecular mass of 85 kDa. Immunolocalization pattern showed some species-specific aspects, such as positive labeling at the equatorial region of the sperm head. Cariporide, a selective NHE1 inhibitor, significantly reduced pHi recovery (85%). Similarly, exposure to cariporide significantly inhibited different motility parameters, including those related to sperm capacitation. In vitro fertilization (IVF) was not affected by cariporide, possibly due to the non-dramatic, although significant, drop in motility and velocity parameters or due to prolonged exposure during IVF, which may have caused progressive loss of its inhibitory effect. In conclusion, this is the first study documenting, in a large animal model (sheep) of well-known translational relevance, a direct functional role of NHE on sperm pHi and motility. The postulated specificity of cariporide toward isoform 1 of the Na+/H+ exchanger seems to suggest that NHE1 may contribute to the observed effects on sperm cell functionality.

Characterization and in vitro differentiation potency of early-passage canine amnion- and umbilical cord-derived mesenchymal stem cells as related to gestational age.

Fetal adnexa are a non-controversial source of mesenchymal stem cells (MSCs) that have high plasticity, a high proliferation rate, and the ability to differentiate towards multiple lineages. MSC populations have been characterized for their stemness and differentiation capabilities; more recent work has focused on MSC selection and on establishing predictable elements to discriminate the cells with the most potential for regenerative medicine. In this study, we cytogenetically and molecularly characterized and followed the in vitro proliferation and differentiation potential of early-passage canine amniotic membrane MSCs (AM-MSCs) and umbilical cord matrix MSCs (UCM-MSCs) isolated from fetuses at early (35-40 days) and late (45-55 days) gestational ages. We found that cells from both fetal gestational ages showed similar features. In all examined cell lines, the morphology of proliferating cells typically appeared fibroblast-like. Population doublings, passaged up to 10 times, increased significantly with passage number. In both cell types, cell viability and chromosomal number and structure were not affected by gestational age at early passages. Passage-3 AM- and UCM-MSCs from both gestational phases also expressed embryonic (POU5F1) and mesenchymal (CD29, CD44) stemness markers, whereas hematopoietic and histocompatibility markers were never found in any sample. Passage-3 cell populations of each cell type were also multipotential as they could differentiate into neurocytes and osteocytes, based on cell morphology, specific stains, and molecular analysis. These results indicated that MSCs retrieved from the UCM and AM in the early and late fetal phases of gestation could be used for canine regenerative medicine.

Catecholamine plasma levels, IFN-γ serum levels and antibodies production induced by rabies vaccine in dogs selected for their paw preference.

To explore the possible role of the sympathetic nervous activity in the asymmetrical crosstalk between the brain and immune system, catecholamine (E, NE) plasma levels, Interferon-γ (IFN-γ) serum levels and production of antibodies induced by rabies vaccine in dogs selected for their paw preference were measured. The results showed that the direction of behavioural lateralization influenced both epinephrine levels and immune response in dogs. A different kinetic of epinephrine levels after immunization was observed in left-pawed dogs compared to both right-pawed and ambidextrous dogs. The titers of antirabies antibodies were lower in left-pawed dogs than in right-pawed and ambidextrous dogs. Similarly, the IFN-γ serum levels were lower in left-pawed dogs than in the other two groups. Taken together, these findings showed that the left-pawed group appeared to be consistently the different group stressing the fundamental role played by the sympathetic nervous system as a mechanistic basis for the crosstalk between the brain and the immune system.

Testicular expression of heat SHOCK proteins 60, 70, and 90 in cryptorchid horses.

Heat shock proteins are the most evolutionarily conserved protein families induced by stressors including hyperthermia. In the context of pathologies of the male reproductive tract, cryptorchidism is the most common genital defect that compromises the reproductive potential of the male because it induces an increase in intratesticular temperature. In equine species, cryptorchidism affects almost 9 % of newborns and few studies have been carried out on the molecular aspects of the retained testis. In this study, the expression pattern of HSP60, 70, and 90 in abdominal and inguinal testes, in their contralateral descended normally testes, and in testes of normal horses were investigated by Western blot and immunohistochemistry. The histomorphological investigation of retained and scrotal testes was also investigated. The seminiferous epithelium of the retained testes showed a vacuolized appearance and displayed a completely blocked spermatogenesis for lacking meiotic and spermiogenetic cells. On the contrary, the contralateral scrotal testes did not show morphological damage and the seminiferous epithelium displayed all phases of the spermatogenetic cycle as in the normal testes. The morphology of Leydig cells was not affected by the cryptorchid state. Western blot and immunohistochemistry evidenced that equine testis (both scrotal and retained) expresses the three investigated HSPs. More in detail, the Western blot evidenced that HSP70 is the more expressed chaperone and that together with HSP90 it is highly expressed in the retained gonad (P < 0.05). The immunohistochemistry revealed the presence of the three HSPs in the spermatogonia of normal and cryptorchid testes. Spermatogonia of retained testes showed the lowest expression of HSP60 and the highest expression of HSP90. Spermatocytes, spermatids of scrotal testes, and the Sertoli cells of retained and scrotal testes did not display HSP60 whereas expressed HSP70 and HSP90. These two proteins were also localized in the nucleus of the premeiotic cells. The Leydig cells displayed the three HSPs with the higher immunostaining of HSP70 and 90 in the cryptorchid testes. The results indicate that the heat stress condition occurring in the cryptorchid testis influences the expression of HSPs.

The abundance and localization of heat shock proteins (HSP)-60, -70, and -90 in the oviductal ampulla of hamadryas baboon (Papio hamadryas) during the menstrual cycle.

The presence of HSPs in female reproductive and their relationship with the steroid hormone fluctuation have been reported in several mammals but not in non-human primates. The present research dealt with the oviductal expression and localization of the more studied HSPs (60, 70, and 90) as well as the morphological changes in the Hamadryas baboon (Papio hamadryas) during the follicular, preovulatory, and luteal phases of the menstrual cycle. Therefore, western blots, histomorphological, and immunohistochemical analyses were carried out. The results of western blot analysis displayed the lowest HSP expression in the luteal phase. The histomorphology showed that the mucosal epithelium consisted of undifferentiated cuboidal cells in follicular and luteal phases and well-distinguishable columnar ciliated and non-ciliated cells during the preovulatory phase. Immunohistochemistry evidenced that the mucosal epithelium contained cytoplasmic and nuclear HSP60, 70, and 90 immunostaining in the follicular and luteal phases. During the preovulatory phase, the non-ciliated cells showed: (i) cytoplasmic HSP60; (ii) nuclear and cytoplasmic HSP90. Ciliated cells showed cytoplasmic and ciliary HSP70 and ciliary HSP90. The stromal cells and myocytes of muscular layer displayed a decreased cytoplasmic HSP60 in the preovulatory phase and nuclear and low cytoplasmic HSP70 throughout the menstrual cycle. Nuclear HSP90 decreased in ampulla stromal cells and the follicular phase myocytes. These findings indicate that the expression pattern of HSP60,70, and 90 is related to the morphofunctional features of the baboon oviductal ampulla during the menstrual cycle and could represent a referent point for further studies in the oviduct of Primates.

Morphology, Glycan Pattern, Heat Shock Proteins, and Sex Steroid Receptors Expression in the Tubal Fimbria Epithelium of the Baboon Papio hamadryas during the Menstrual Cycle.

The oviductal fimbria is the first extraovarian anatomical structure that the cumulus-oocyte complex (COC) encounters, and is sensitive to sex hormone changes. The morphology, glycan pattern, expression of heat shock proteins (HSPs), estradiol receptor (ER), and progesterone receptor (PR) were investigated in the oviductal fimbria epithelium of the baboon (Papio hamadryas) during the menstrual cycle. The morphology was investigated by light and scanning electron microscopy; the glycopattern was characterized using conventional and lectin histochemistry; HSPs (60, -70, -90), ER, and PR were localized immunohistochemically. Well-differentiated ciliated and nonciliated cells were present only during the preovulatory phase. The nonciliated cells contained small apical protrusions and thin microvilli. During the preovulatory phase (1) the luminal surface of the fimbria displayed acidic glycans, complex N-glycans containing fucose, and oligolactosamine residues; (2) nonciliated cells expressed HSP60 and HSP90 in the apical blebs, HSP70 in the nucleus and cytoplasm, as well as nuclear ERα and PR; (3) ciliated cells showed HSP70 in the nucleus, cytoplasm, and cilia that also expressed HSP90 and PR. These results are related to the function of the fimbria where the early COC-oviduct crosstalk occurs and may represent a benchmark for translational studies of other primates.

Relationship between Fat Status, Stage of Gonadal Maturity and Hormonal Variation of Turdus philomelos (C.L. Brehm, 1831) Wintering in Apulia during 2018-2020.

In this study, we aimed to evaluate the development of the fattening condition and the reproductive status of the song thrush from December to February. For this purpose, the chemical and fatty acid compositions of the pectoral muscle were analyzed in relation to the fattening state of the birds. Moreover, their reproductive activity was evaluated via the anatomical and pathological examination of tissues and through the assessment of sex steroid profiles. One hundred ninety-five thrushes captured by local hunters during the 2018-2019 and 2019-2020 hunting seasons in different provinces of the Apulia region in Italy were used. The first step was the measurement of bird body mass, and the amount of subcutaneous body fat was estimated visually. During post-mortem examinations, the pectoral muscle was excised and used for chemical and fatty acid analysis and a hormone assay, respectively. Moreover, ovaries and testicles were evaluated to determine the degree of maturation and thus the reproductive status of the birds. The results regarding fattening status and fatty acid profile confirmed that in January-February, thrushes change their diet, increasing their intake of oleic acid, likely to better cope with low temperatures and prepare for long-distance migration. In both male and female thrushes, the concentrations of sex hormones confirmed a phase of reproductive quiescence from December to February, which was also confirmed through histological examination of the gonads.

Male infertility and copy number variants (CNVs) in the dog: a two-pronged approach using Computer Assisted Sperm Analysis (CASA) and Fluorescent In Situ Hybridization (FISH).

BACKGROUND: Infertility affects ~10-15% of couples trying to have children, in which the rate of male fertility problems is approximately at 30-50%. Copy number variations (CNVs) are DNA sequences greater than or equal to 1 kb in length sharing a high level of similarity, and present at a variable number of copies in the genome; in our study, we used the canine species as an animal model to detect CNVs responsible for male infertility. We aim to identify CNVs associated with male infertility in the dog genome with a two-pronged approach: we performed a sperm analysis using the CASA system and a cytogenetic-targeted analysis on genes involved in male gonad development and spermatogenesis with fluorescence in situ hybridization (FISH), using dog-specific clones. This analysis was carried out to evaluate possible correlations between CNVs on targeted genes and spermatogenesis impairments or infertility factors. RESULTS: We identified two genomic regions hybridized by BACs CH82-321J09 and CH82-509B23 showing duplication patterns in all samples except for an azoospermic dog. These two regions harbor two important genes for spermatogenesis: DNM2 and TEKT1. The genomic region encompassed by the BAC clone CH82-324I01 showed a single-copy pattern in all samples except for one dog, assessed with low-quality sperm, displaying a marked duplication pattern. This genomic region harbors SOX8, a key gene for testis development. CONCLUSION: We present the first study involving functional and genetic analyses in male infertility. We set up an extremely reliable analysis on dog sperm cells with a highly consistent statistical significance, and we succeeded in conducting FISH experiments on sperm cells using BAC clones as probes. We found copy number differences in infertile compared with fertile dogs for genomic regions encompassing TEKT1, DNM2, and SOX8, suggesting those genes could have a role if deleted or duplicated with respect to the reference copy number in fertility biology. This method is of particular interest in the dog due to the recognized role of this species as an animal model for the study of human genetic diseases and could be useful for other species of economic interest and for endangered animal species.

Expression and localization of the mu-opioid receptor (MOR) in the equine cumulus-oocyte complex and its involvement in the seasonal regulation of oocyte meiotic competence.

The micro-opioid receptor (MOR) was identified in equine oocytes, cumulus and granulosa cells. By RT-PCR, a 441bp fragment was observed. By immunoblotting, a 65 kDa band was detected in samples of winter anestrous whereas in cells recovered in breeding season, two bands, 65 and 50 kDa, were found. The 65 kDa band was significantly more intense in winter anestrous specimens. In samples recovered in the breeding season, this band significantly decreased with the raise of follicle size and was heavier in compact oocytes and cumulus cells. The protein was localized on the oolemma and within the cytoplasm of oocytes and cumulus cells. In vitro oocyte maturation rate (MR), analyzed by confocal microscopy for nuclear chromatin, microfilaments and microtubules, was reduced after the addition of 3 x 10(-8) M beta-endorphin in medium without additional hormones. Inhibitory effects of 10(-3) M Naloxone in oocytes collected in anestrous and spring transition were observed, both in presence and absence of hormones added to culture medium. Increased MRs were observed in oocytes collected in anestrous and cultured in presence of 10(-8) M Naloxone. The exposure to 10(-3) M Naloxone induced significant intracellular calcium increases in cumulus cells recovered all over the year. beta-Endorphin 3 x 10(-8) M induced significant calcium increases only in cumulus cells recovered in fall transition and anestrous. Naloxone 10(-8) M did not induce intracellular calcium modifications. We conclude that the MOR is differentially expressed in equine cumulus-oocyte complexes in the different seasons of the year and plays a role in the seasonal regulation of meiotic competence of equine oocytes.

Functional expression of the extracellular calcium sensing receptor (CaSR) in equine umbilical cord matrix size-sieved stem cells.

BACKGROUND: The present study investigates the effects of high external calcium concentration ([Ca(2+)](o)) and the calcimimetic NPS R-467, a known calcium-sensing receptor (CaSR) agonist, on growth/proliferation of two equine size-sieved umbilical cord matrix mesenchymal stem cell (eUCM-MSC) lines. The involvement of CaSR on observed cell response was analyzed at both the mRNA and protein level. METHODOLOGY/PRINCIPAL FINDINGS: A large (>8 µm in diameter) and a small (<8 µm) cell line were cultured in medium containing: 1) low [Ca(2+)](o) (0.37 mM); 2) high [Ca(2+)](o) (2.87 mM); 3) NPS R-467 (3 µM) in presence of high [Ca(2+)](o) and 4) the CaSR antagonist NPS 2390 (10 µM for 30 min.) followed by incubation in presence of NPS R-467 in medium with high [Ca(2+)](o). Growth/proliferation rates were compared between groups. In large cells, the addition of NPS R-467 significantly increased cell growth whereas increasing [Ca(2+)](o) was not effective in this cell line. In small cells, both higher [Ca(2+)](o) and NPS R-467 increased cell growth. In both cell lines, preincubation with the CaSR antagonist NPS 2390 significantly inhibited the agonistic effect of NPS R-467. In both cell lines, increased [Ca(2+)](o) and/or NPS R-467 reduced doubling time values.Treatment with NPS R-467 down-regulated CaSR mRNA expression in both cell lines. In large cells, NPS R-467 reduced CaSR labeling in the cytosol and increased it at cortical level. CONCLUSIONS/SIGNIFICANCE: In conclusion, calcium and the calcimimetic NPS R-467 reduce CaSR mRNA expression and stimulate cell growth/proliferation in eUCM-MSC. Their use as components of media for eUCM-MSC culture could be beneficial to obtain enough cells for down-stream purposes.

Mitochondrial distribution patterns in canine oocytes as related to the reproductive cycle stage.

This study investigates the mitochondrial (mt) distribution in canine ovarian oocytes examined at recovery time, as related to the reproductive cycle stage, and in oviductal oocytes. Ovarian Germinal Vesicle (GV) stage oocytes were recovered from bitches in anestrous (A, n=2), follicular phase (F, n=4), ovulation (O, n=2), early luteal (EL, n=7) and mid/late luteal phase (MLL, n=2). Oviductal GV, metaphase I (MI) or MII stage oocytes were recovered from six bitches between 56 and 110 h after ovulation. Mitochondria were revealed by using MitoTracker Orange CMTM Ros and confocal microscopy. In ovarian oocytes, three mt distribution patterns were found: (I) small aggregates diffused throughout the cytoplasm; (II) diffused tubular networks; (III) pericortical tubular networks. Significantly higher rates of oocytes showing heterogeneous mt patterns (II+III) were obtained from bitches in F (75%) and in O (96%) compared with bitches in A (31%; F vs. A: P<0.05; O vs. A: P<0.001), in EL (61%; O vs. EL: P<0.01), or in MLL (0%; F vs. MLL: P<0.05; O vs. MLL: P<0.001). Fluorescence intensity did not vary according to mt distribution pattern except that it was lower in oocytes recovered in EL phase and showing small mt aggregations (P<0.001). The majority of ovulated MII stage oocytes (79%) showed diffused tubular mt network. We conclude that mt distribution pattern of canine ovarian immature oocytes changes in relation to reproductive cycle stage and that patterns observed in oocytes recovered from bitches in periovulatory phases are heterogeneous and similar to those of in vivo matured oocytes.

Expression and immunolocalization of the mu-opioid receptor in human sperm cells.

The expression of the mu-opioid receptor in human sperm cells was investigated by immunocytochemistry and immunoblot analyses. Results demonstrated that the receptor is localized on the acrosomal region and the neck portion of the sperm head. These results suggest a possible role for the mu-opioid receptor in mediating the action of endogenous opioid peptides on sperm cells during the complex and poorly characterized cellular events that enable spermatozoa to fertilize oocytes.

Expression and subcellular localization of the mu-opioid receptor in equine spermatozoa: evidence for its functional role.

The development of fertilizing ability in sperm cells is associated with changes in the plasma membrane. However, to date the exact nature of sequentially activated primary receptors and channels and the signal transduction pathways derived from these remains elusive. We analyzed the expression and localization of the mu-opioid receptor in equine spermatozoa. A transcript corresponding to the third extracellular loop that selectively binds mu agonists was amplified, sequenced and compared with the known sequences in humans, rats and cattle. The amplification product showed a high degree of nucleotide conservation. By immunofluorescence, mu-opioid receptor labeling was found on the sperm head and on the tail and disappeared in the acrosomal region of acrosome-reacted sperm cells. Immunoblotting revealed two bands of 50 and 65 kDa. Effects of the opioid antagonist naloxone on motility and on viability and capacitation/acrosome reaction were investigated by computer-assisted sperm analysis and Hoechst 33258/chlortetracycline (H258/CTC) staining. Progressive motility was significantly reduced after 3 h incubation in 10(-3) M naloxone (P <0.05), whereas it increased significantly after 5 h in 10(-8) M naloxone (P <0.05). Sperm velocity at 5 h was significantly reduced by the addition of 10(-3) M naloxone (P <0.05), but increased significantly in the presence of 10(-8) M (P <0.001). Curvilinear velocity and amplitude of lateral head displacement in spermatozoa incubated in the presence of naloxone were not indicative of hyperactivation. H258/CTC staining showed that 10(-8) M naloxone significantly stimulated capacitation (P <0.01) after 3 h. However, it had no effect on sperm cell viability and acrosomal status. Overall, this study provides the first evidence that the mu-opioid receptor is expressed in equine spermatozoa and that naloxone significantly affects motility and capacitation.