Haemodynamic changes are involved in the cyclic remodelling of ovarian tissue that occurs during final follicular growth, ovulation and new corpus luteum development. The aim of this study was to characterize the real-time changes in the blood flow within the follicle wall associated with the LH surge, ovulation and corpus luteum development in cows. Normally cyclic cows with a spontaneous ovulation (n = 5) or a GnRH-induced ovulation (n = 5) were examined by transrectal colour and pulsed Doppler ultrasonography to determine the area and the time-averaged maximum velocity (TAMXV) of the blood flow within the preovulatory follicle wall and the early corpus luteum. Ultrasonographic examinations began 48 h after a luteolytic injection of PGF2α analogue was given at the mid-luteal phase of the oestrous cycle. Cows with spontaneous ovulation were scanned at 6 h intervals until ovulation occurred. Cows with GnRH-induced ovulation were scanned just before GnRH injection (0 h), thereafter at 0.5, 1, 2, 6, 12, 24 h and at 24 h intervals up to day 5. Blood samples were collected at the same time points for oestradiol, LH and progesterone determinations. Cows with both spontaneous and GnRH-induced ovulation showed a clear increase in the plasma concentration of LH (LH surge) followed by ovulation 26-34 h later. In the colour Doppler image of the preovulatory follicle, the blood flow before the LH surge was detectable only in a small area in the base of the follicle. An acute increase in the blood flow velocity (TAMXV) was detected at 0.5 h after GnRH injection, synchronously with the initiation of the LH surge. At 12 h after the LH surge, the plasma concentrations of oestradiol decreased to basal concentrations. The TAMXV remained unchanged after the initial increase until ovulation, but decreased on day 2 (12-24 h after ovulation). In the early corpus luteum, the blood flow (area and TAMXV) gradually increased in parallel with the increase in corpus luteum volume and plasma progesterone concentration from day 2 to day 5, indicating active angiogenesis and normal luteal development. Collectively, the complex structural, secretory and functional changes that take place in the ovary before ovulation are closely associated with a local increase in the blood flow within the preovulatory follicle wall. The result of the present study provides the first visual information on vascular and blood flow changes associated with ovulation and early corpus luteum development in cows. This information may be essential for future studies involving pharmacological control of blood flow and alteration of ovarian function.

Recent studies suggest that IGF-I is a crucial regulatory factor in follicular growth during early post-partum period. The aim of the present study was to determine in detail the changing profiles of metabolic and reproductive hormones in relation to ovulation of the dominant follicle (DF) of the first follicular wave post-partum in high-producing dairy cows. Plasma concentrations of related hormones in 22 multiparous Holstein cows were measured from 4 weeks pre-partum to 3 weeks post-partum, and the development of DF was observed with colour Doppler ultrasound. Thirteen cows showed ovulation by 15.2 days post-partum. Anovulatory cows showed higher GH and lower IGF-I levels than those in ovulatory cows during the peri-partum period. Each DF developed similarly, and a clear blood flow in the follicle wall was observed despite ovulation or anovulation. In addition, detailed endocrine profiles were analyzed in 9 out of the 22 cows. Five cows showed an increase in plasma oestradiol-17β (E2) with follicular growth followed by E2 peak, LH surge and ovulation. In these cows, plasma IGF-I concentrations remained high until 10 days post-partum followed by a gradual decrease. Subsequently, the insulin level increased together with the E2 peak towards ovulation. These profiles were not observed in anovulatory cows. In conclusion, our data strongly support the concept that IGF-I and insulin represent ‘metabolic signals’ of the resumption of ovarian function post-partum in high-producing dairy cows. Moreover, we provide the first visual evidence that both ovulatory and anovulatory DFs of the first follicular wave post-partum are similarly supplied with active blood flow. Abstract Recent studies suggest that IGF-I is a crucial regulatory factor in follicular growth during early post-partum period. The aim of the present study was to determine in detail the changing profiles of metabolic and reproductive hormones in relation to ovulation of the dominant follicle (DF) of the first follicular wave post-partum in high-producing dairy cows. Plasma concentrations of related hormones in 22 multiparous Holstein cows were measured from 4 weeks pre-partum to 3 weeks post-partum, and the development of DF was observed with colour Doppler ultrasound. Thirteen cows showed ovulation by 15.2 days post-partum. Anovulatory cows showed higher GH and lower IGF-I levels than those in ovulatory cows during the peri-partum period. Each DF developed similarly, and a clear blood flow in the follicle wall was observed despite ovulation or anovulation. In addition, detailed endocrine profiles were analyzed in 9 out of the 22 cows. Five cows showed an increase in plasma oestradiol-17β (E2) with follicular growth followed by E2 peak, LH surge and ovulation. In these cows, plasma IGF-I concentrations remained high until 10 days post-partum followed by a gradual decrease. Subsequently, the insulin level increased together with the E2 peak towards ovulation. These profiles were not observed in anovulatory cows. In conclusion, our data strongly support the concept that IGF-I and insulin represent ‘metabolic signals’ of the resumption of ovarian function post-partum in high-producing dairy cows. Moreover, we provide the first visual evidence that both ovulatory and anovulatory DFs of the first follicular wave post-partum are similarly supplied with active blood flow.

Background: After ovulation in the cow, the corpus luteum (CL) rapidly develops within a few days with angiogenesis and progesterone production. CL formation resembles an inflammatory response due to the influx of immune cells. Neutrophils play a role in host defense and inflammation, and secrete chemoattractants to stimulate angiogenesis. We therefore hypothesized that neutrophils infiltrate in the developing CL from just after ovulation and may play a role in angiogenesis of the CL. Methods and Results: Polymorphonuclear neutrophils (PMN) were detected in CL tissue by Pas-staining, and interleukin-8 (IL-8, a neutrophil-specific chemoattractant) was measured in supernatant of the CL tissue culture: considerable amounts of PMNs and the high level of IL-8 were observed during the early luteal phase (days 1-4 of the estrous cycle). PMNs and IL-8 were low levels in the mid and late luteal phases, but IL-8 was increased during luteal regression. The PMN migration in vitro was stimulated by the supernatant from the early CL but not from the mid CL, and this activity was inhibited by neutralizing with an anti-IL-8 antibody, indicating the major role of IL-8 in inducing active PMN migration in the early CL. Moreover, IL-8 stimulated proliferation of CL-derived endothelial cells (LECs), and both the supernatant of activated PMNs and IL-8 stimulated formation of capillary-like structures of LECs. Conclusion: PMNs migrate into the early CL partially due to its major chemoattractant IL-8 produced at high levels in the CL, and PMNs is a potential regulator of angiogenesis together with IL-8 in developing CL in the cow.

This study aimed to investigate the presence of polymorphonuclear neutrophils (PMNs) in bovine oviduct fluid under physiological conditions and to determine the possible role of bovine oviduct epithelial cells (BOECs) in the regulation of the phagocytic activity of PMNs for sperm. During the pre-ovulatory stage, PMNs were identified in the bovine oviduct fluid in relatively constant numbers. In our experiments, PMNs were incubated for 4 h with the supernatant of cultured BOECs stimulated for 24 h by LH (10 ng/ml). Phagocytosis was then assayed by co-incubation of these PMNs with sperm treated to induce capacitation. The BOEC supernatant significantly suppressed sperm phagocytosis by PMNs, and the LH-stimulated BOEC supernatant further suppressed phagocytosis. Importantly, in the BOEC culture, LH stimulated the secretion of prostaglandin E₂ (PGE₂), which dose-dependently (10⁻⁶, 10⁻⁷, and 10⁻⁸ M) suppressed sperm phagocytosis by PMNs. Furthermore, a PGEP₂ receptor antagonist significantly abrogated the inhibition of phagocytosis by the LH-stimulated BOEC supernatant. Additionally, using scanning electron microscopy, incubation of PMNs with either PGE₂ or LH-stimulated BOEC supernatant before phagocytosis was found to prevent the formation of DNA-based neutrophil extracellular traps for sperm entanglement. The results indicate that sperm are exposed to PMNs in the oviduct and PGE₂ released into the oviduct fluid after LH stimulation may play a major role in the suppression of the phagocytic activity of PMNs for sperm via interaction with EP₂ receptors. Thus, the bovine oviduct provides a PGE₂-rich microenvironment to protect sperm from phagocytosis by PMNs, thereby supporting sperm survival in the oviduct. Free Japanese abstract A Japanese translation of this abstract is freely available at http://www.reproduction-online.org/content/147/2/211/suppl/DC1 Abstract This study aimed to investigate the presence of polymorphonuclear neutrophils (PMNs) in bovine oviduct fluid under physiological conditions and to determine the possible role of bovine oviduct epithelial cells (BOECs) in the regulation of the phagocytic activity of PMNs for sperm. During the pre-ovulatory stage, PMNs were identified in the bovine oviduct fluid in relatively constant numbers. In our experiments, PMNs were incubated for 4 h with the supernatant of cultured BOECs stimulated for 24 h by LH (10 ng/ml). Phagocytosis was then assayed by co-incubation of these PMNs with sperm treated to induce capacitation. The BOEC supernatant significantly suppressed sperm phagocytosis by PMNs, and the LH-stimulated BOEC supernatant further suppressed phagocytosis. Importantly, in the BOEC culture, LH stimulated the secretion of prostaglandin E₂ (PGE₂), which dose-dependently (10⁻⁶, 10⁻⁷, and 10⁻⁸ M) suppressed sperm phagocytosis by PMNs. Furthermore, a PGEP₂ receptor antagonist significantly abrogated the inhibition of phagocytosis by the LH-stimulated BOEC supernatant. Additionally, using scanning electron microscopy, incubation of PMNs with either PGE₂ or LH-stimulated BOEC supernatant before phagocytosis was found to prevent the formation of DNA-based neutrophil extracellular traps for sperm entanglement. The results indicate that sperm are exposed to PMNs in the oviduct and PGE₂ released into the oviduct fluid after LH stimulation may play a major role in the suppression of the phagocytic activity of PMNs for sperm via interaction with EP₂ receptors. Thus, the bovine oviduct provides a PGE₂-rich microenvironment to protect sperm from phagocytosis by PMNs, thereby supporting sperm survival in the oviduct. Free Japanese abstract A Japanese translation of this abstract is freely available at http://www.reproduction-online.org/content/147/2/211/suppl/DC1

Sperm are allogenic to the female genital tract; however, oviducts provide optimal conditions for survival and capacitation of these non-self cells until fertilization. Recently, we showed that oviduct-conditioned media and prostaglandin E₂ (PGE₂) suppress sperm phagocytosis by polymorphonuclear neutrophils (PMNs) under physiological conditions. We hypothesized that sperm binding to bovine oviduct epithelial cells (BOECs) could change the local innate immunity via PGE₂. As the first step to obtain basic information, sub-confluent BOEC monolayers were co-cultured with swim-up sperm for 2 h. BOECs with viable bound sperm were cultured for an additional 3, 6, 12, or 24 h. Then, we confirmed the impact of the sperm-BOEC binding on both BOECs and PMN gene expression. Immunohistochemistry revealed that BOECs strongly express TGFB1 and IL10 in the oviduct. Sperm binding to BOECs in culture induced the anti-inflammatory cytokines (TGFB1 and IL10) and PGE₂ production by BOECs. Exogenous PGE₂ in vitro suppressed pro-inflammatory cytokine expression (TNF and IL1B) in BOECs. Moreover, pre-exposure of PMNs to BOEC-conditioned media suppressed the TNF expression, but the BOEC media co-cultured with sperm stimulated PMNs to express TGFB1 and IL10, with increasing PGE₂ secretion. Of note, exogenous PGE₂ led PMNs in vitro to decrease their TNF expression and increase anti-inflammatory cytokines expression. Our findings strongly suggest that BOECs provide an anti-inflammatory environment under physiological conditions and the sperm-BOEC binding further strengthens this milieu thus suppresses PMNs in the bovine oviduct. PGE₂ is likely to drive this stable anti-inflammatory environment in the oviduct.