Physiology and endocrinology symposium: evidence that oviduct secretions influence sperm function: a retrospective view for livestock

The mammalian oviduct has long been recognized as an organ essential for successful reproduction. Bovine, ovine, porcine, and equine animal models have offered clear advantages for oviduct study related to gamete physiology, fertilization, and early embryonic development. Livestock species are amenable to surgical alteration of the reproductive tract, estrous cycle manipulation, gamete cryopreservation, and AI, as well as in vitro fertilization and embryo production. Although most reproductive technology developed for livestock was intended to benefit production animal agriculture, these techniques are a treasure trove of tools for researchers to better understand how the oviduct influences gamete function. Oviduct secretions obtained from in vitro tissue cultures or via indwelling oviduct catheters have been used for analyses to define the protein, lipid, carbohydrate, enzyme, and electrolyte compositions of the secretions during the estrous cycle or in response to hormone treatment. Oviduct secretions or components purified from them have also been used in in vitro assays to assess their ability to bind to sperm, influence sperm viability, motility, sperm capacitation, the acrosome reaction, sperm-egg binding, and egg penetration, as well as subsequent embryonic development. Compelling data have emerged which show that the composition of secretions differs during the estrous cycle and that their composition differs whether they originate from the ampullary or isthmic regions of the oviduct. These differences in composition are functionally relevant and associated with different responses by sperm. Evidence indicatess that oviduct-specific glycoproteins, glycosaminoglycans, carbohydrates, norepinepherine, catecholamines, heat-shock protein, and osteopontin are components of the oviductal milieu that have the capacity to modulate sperm function. Future research on the livestock oviduct will likely define the role that oviduct secretions have in modulating sperm function and how these modifications ultimately affect fertilization and embryo development.     

An Acute-phase Protein as a Regulator of Sperm Survival in the Bovine Oviduct: Alpha 1-acid-glycoprotein Impairs Neutrophil Phagocytosis of Sperm In Vitro

We have previously shown that polymorphonuclear neutrophils (PMNs) are present in bovine oviduct fluid under physiological conditions, and that the oviduct provides a microenvironment that protects sperm from phagocytosis by PMNs. Alpha 1-acid glycoprotein (AGP) is a major acute-phase protein produced mainly in the liver that has immunomodulatory functions. AGP mRNA is expressed in extrahepatic organs, such as the lung, kidney, spleen, lymph node, uterus, and ovary. Therefore, in this study, we investigated, 1) the local production of AGP in the bovine oviduct, 2) the effect of AGP on the phagocytic activity of PMNs for sperm and superoxide production and 3) the impact of AGP desialylation on the PMN phagocytosis of sperm. The AGP gene was expressed in cultured bovine oviduct epithelial cells (BOECs) and AGP protein was detected in oviduct fluid. Preexposure of PMNs to AGP at physiological levels impaired PMN phagocytosis for sperm and superoxide generation. The desialylation of AGP eliminated these suppressive effects of AGP on PMN. Scanning electron microscopy revealed that AGP drastically reduced the formation of DNA-based neutrophil extracellular traps (NETs) for sperm entanglement. Additionally, AGP dose-dependently stimulated BOECs to produce prostaglandin E₂ (PGE₂) which has been shown to partially contribute to the regulation of sperm phagocytosis in the bovine oviduct. AGP and PGE₂ at concentrations detected in the oviducts additively suppressed sperm phagocytosis by PMNs. These results provide evidence that locally produced AGP may be involved in protecting sperm from phagocytosis by PMNs in the bovine oviduct.     

Hyperactivated Motility in Sperm

Hyperactivation is a movement pattern seen in sperm at the site and time of fertilization in mammals. It may be critical to the success of fertilization, because it enhances the ability of sperm to detach from the wall of the oviduct, to move around in the labyrinthine lumen of the oviduct, to penetrate mucous substances and, finally, to penetrate the zona pellucida of the oocyte. The movement of hyperactivated sperm appears different under different physical conditions and in different species, but basically it involves an increase in flagellar bend amplitude and, usually, beat asymmetry. Presumably, a signal or signals exist in the oviduct to initiate hyperactivation at the appropriate time; however, none has yet been identified with certainty. While the signal transduction cascade regulating hyperactivation remains to be completely described, it is clear that calcium ions interact with the axoneme of the flagellum to switch on hyperactivation. Although hyperactivation often occurs during the process of capacitation, the two events are regulated by somewhat different pathways.     

钙离子浓度对绵羊精子获能的影响

为探讨钙离子浓度对绵羊精子获能的影响,将绵羊精子在不同钙离子浓度的绵羊输卵管合成液（SOF液）中培养,并用钙离子载体A23187进行诱导。结果发现,不同钙离子浓度对绵羊精子的影响差别不明显,经A23187诱导后,1.7 mmol/L组和2 mmol/L组的顶体反应率（AR）与其他组有显著差异（P<0.05）,1.7 mmol/L浓度中,绵羊精子存活时间为16 h,2 mmol/L浓度中的存活时间为10 h,因此认为绵羊精子获能的适宜钙离子浓度为1.7 mmol/L,且获能过程中钙离子的存在是必要的。     

Oviducal storage of spermatozoa in the turkey: its relevance to artificial insemination technology

1. This paper addresses questions concerning the role of oviducal sperm selection and storage in the relative inefficiency of turkey reproduction. 2. Before the onset of egg production, the turkey has the biological potential to store spermatozoa in its oviduct for periods up to 5 weeks; after the onset of production fewer spermatozoa are retained by the oviduct. 3. A better understanding of the biological mechanisms which regulate sperm selection and storage in the turkey oviduct is necessary to make significant advances in artificial insemination technology.     

Addition of oxytocin to semen extender improves both sperm transport to the oviduct and conception rates in pigs following AI

Oxytocin (OXT) contained in boar semen is known to produce uterine contraction; therefore, we hypothesized that the co‐injection of OXT with sperm would improve artificial insemination (AI) using liquid or frozen‐thawed boar sperm. We initially examined whether OXT added to semen extender improved sperm transport to the oviduct. Although the addition of OXT did not affect the fresh or frozen‐thawed sperm motility or acrosomal integrity, it significantly increased the number of sperm in the oviduct at 6 h after AI injection with OXT, as compared with the control (P < 0.05). Moreover, some sperm were observed in the sperm reservoir of the isthmus in the OXT treatment group, whereas few sperm were observed in the control. When OXT was added to the semen extender immediately prior to AI, the conception rates were significantly higher in both fresh semen and frozen‐thawed semen than in the control group (P < 0.05: liquid, 87.5% vs. 70.5%; frozen‐thawed, 89.8% vs. 75.0%). From these results, we concluded that the addition of OXT to the semen extender assisted in sperm transportation from the uterus to the oviduct, which resulted in improved reproductive performance.     

Fertilization and Early Embryonic Development in the Porcine Fallopian Tube

Fertilization and early embryo development relies on a complex interplay between the Fallopian tube and the gametes before and after fertilization. Thereby the oviduct, as a dynamic reproductive organ, enables reception, transport and maturation of male and female gametes, their fusion, and supports early embryo development. This paper reviews current knowledge regarding physiological processes behind the transport of boar spermatozoa, their storage in and release from the functional sperm reservoir (SR), and of the interactions that newly ovulated oocytes play within the tube during their transport to the site of fertilization. Experimental evidence of an ovarian control on sperm release from the SR is highlighted. Furthermore, the impact of oviductal secretion on sperm capacitation, oocyte maturation, fertilization and early embryo development is stressed.     

家禽精子迁移率Mobility是反映家禽精液质量的重要指标

精子活率是精子的重要特性,传统精子活率的评价方法并不代表精子在家禽机体内生殖道的真实运动情况,具有主观性的特点,准确性差。精子迁移率Mobility的测定方法模拟精子在母鸡体内液体环境和温度下的运动情况,能够反映精子在体内真正的活力。从37只纯种白洛克公鸡中挑选出具有不同精子迁移率Mobility的高、低2组,每组5只公鸡,其精子迁移率分别为1.103,0.219,相应的受精率分别为83.7%、76.8%(P<0.05)。结果表明:精子迁移率Mobility反映了不同家禽个体精液质量的差异,是预测人工授精效果的一个重要指标。     

Development of an in vitro oviduct epithelial explants model for studying sperm–oviduct binding in the buffalo

In this study, we developed an in vitro model for studying sperm–oviduct binding in the buffalo. Oviduct explants were prepared by overnight culture of epithelial cells in TCM‐199 medium under 5% CO₂ at 38.5 °C. Cryopreserved spermatozoa from buffalo bulls (n = 4) were incubated with the oviduct explants, and the sperm–oviduct explants complex was stained with JC‐1. The effect of sperm concentration (2, 3 and 4 million), size of the oviduct explants (<0.2, 0.2–0.3, 0.3–0.4 and >0.4 mm²) and time of incubation (1 hr and 4 hr) on binding index (BI—number of sperm bound to unit area of explants) was studied. No significant difference was observed in the BI among <0.2, 0.2–0.3 and 0.3–0.4 mm² size of explants; however, the BI decreased significantly (p < .05) when the size of explants exceeded 0.4 mm². The BI decreased significantly (p < .05) when the sperm concentration was increased to 4 million, while the duration of incubation did not have any significant effect on the BI. The interaction of bulls with explants size, sperm concentration and incubation time was not significant. The developed assay has the potential to be used as an in vitro model for studying sperm–oviduct binding in the buffalo.     

Experimental evidence for the influence of cumulus-oocyte-complexes on sperm release from the porcine oviductal sperm reservoir

In the pig, a temporal relationship is suggested between sperm release from the sperm reservoir (SR) and ovulation, but the mechanism(s) is still under discussion. In two experiments, the influence of transferred ova on the release of SR-spermatozoa at ovulation and the effect of supplementation with non-sulfated glycosaminoglycan hyaluronan (HA) on embryo development and the number of accessory spermatozoa, respectively, were examined. PMSG/hCG primed ovectomized gilts that had previously received endoscopic low-dose insemination into the cranial uterine horn were used as an experimental model. After salpingectomy, tubal segments (ampulla, cranial, and caudal isthmus) were flushed and sperm numbers or respective accessory spermatozoa were counted. In Experiment 1, the distribution of the sperm population was altered in the presence of cumulus-oocyte-complexes (COCs). A higher proportion of spermatozoa was found after transfer of COCs into one oviduct in the ampulla and cranial isthmus segments compared with the controls (17.5 vs. 4.9%, p<0.05). In Experiment 2, the quality of the transferred ova and treatment influenced the presence of accessory spermatozoa. Transfer of COCs together with HA increased (p<0.05) the number of accessory spermatozoa compared with the other treatment groups and was similar to those in the "undisturbed" controls. No modifications were obtained regarding mean blastomere numbers (2.6 +/- 0.2 to 3.1 +/- 0.2). In summary, this study was demonstrated that cumulus-oocyte-complexes may be involved in triggering sperm release from the pig oviductal SR and that HA might be related to sperm release.     

The porcine sperm reservoir in relation to the function of hyaluronan

The oviduct plays a role in successful animal reproduction not only in spermatozoa and ova transport to the fertilization site but also by affording a microenvironment for fertilization and early embryonic development. The sperm reservoir (SR) is restricted in the uterotubal junction (UTJ) and caudal isthmus. Billions of porcine spermatozoa are distributed to the female reproductive tract during/after insemination, and small amounts of them are stored for about 36–40 hours in the SR, which maintains sperm viability in the pre-ovulation period through its surface epithelium and production of fluid. The SR regulates the release of spermatozoa so that only a small population moves towards the fertilization site (ampulla) to decrease polyspermy. This review attempts to provide information about the structure and function of the porcine SR, its intraluminal content (hyaluronan, HA), and the influences of HA on porcine spermatozoa in vivo. In pigs, the spermatozoa are stored in a mucous-like fluid within the UTJ and caudal isthmus in the pre-ovulation period. The oviduct fluid contains sulfated glycosaminoglycans (GAGs) and non-sulfated GAGs, i.e., HA. It is interesting to note that HA is synthesized by hyaluronan synthase-3 (HAS-3), and its receptor, CD44, is found in the epithelium of the porcine SR site. Additionally, sperm capacitation does not occur in vivo in the SR during the pre- and peri-ovulation periods, but spermatozoa in the SR will attempt to capacitate if exposed to bicarbonate. However, capacitation in the SR will rise in the post-ovulation period, indicating the role of HA in modulating sperm capacitation after ovulation. All data support the understanding that the porcine SR ensures the viability of fertile spermatozoa and maintains the non-capacitated status during the pre-ovulation period. This basic knowledge about the SR is believed to be useful to advance sperm preparation procedures for in vitro fertilization (IVF) and improve the preservation process of porcine semen.     

Number of Spermatozoa in the Crypts of the Sperm Reservoir at About 24 h After a Low‐Dose Intrauterine and Deep Intrauterine Insemination in Sows

The aim of this study was to investigate the number of spermatozoa in the crypts of the utero‐tubal junction (UTJ) and the oviduct of sows approximately 24 h after intrauterine insemination (IUI) and deep intrauterine insemination (DIUI) and compared with that of conventional artificial insemination (AI). Fifteen crossbred Landrace × Yorkshire (LY) multiparous sows were used in the experiment. Transrectal ultrasonography was performed every 4 h to examine the time of ovulation in relation to oestrous behaviour. The sows were inseminated with a single dose of diluted fresh semen by the AI (n = 5), IUI (n = 5) and DIUI (n = 5) at approximately 6–8 h prior to the expected time of ovulation, during the second oestrus after weaning. The sperm dose contained 3000 × 10⁶ spermatozoa in 100 ml for AI, 1,000 × 10⁶ spermatozoa in 50 ml for IUI and 150 × 10⁶ spermatozoa in 5 ml for DIUI. The sows were anaesthetized and ovario‐hysterectomized approximately 24 h after insemination. The oviducts and the proximal part of the uterine horns (1 cm) on each side of the reproductive tracts were collected. The section was divided into four parts, i.e. UTJ, caudal isthmus, cranial isthmus and ampulla. The spermatozoa in the lumen in each part were flushed several times with phosphate buffer solution. After flushing, the UTJ and all parts of the oviducts were immersed in a 10% neutral buffered formalin solution. The UTJ and each part of the oviducts were cut into four equal parts and embedded in a paraffin block. The tissue sections were transversely sectioned to a thickness of 5 μm. Every fifth serial section was mounted and stained with haematoxylin and eosin. The total number of spermatozoa from 32 sections in each parts of the tissue (16 sections from the left side and 16 sections from the right side) was determined under light microscope. The results reveal that most of the spermatozoa in the histological section were located in groups in the epithelial crypts. The means of the total number of spermatozoa in the sperm reservoir (UTJ and caudal isthmus) were 2296, 729 and 22 cells in AI, IUI and DIUI groups, respectively (p < 0.01). The spermatozoa were found on both sides of the sperm reservoir in all sows in the AI and the IUI groups. For the DIUI group, spermatozoa were not found on any side of the sperm reservoir in three out of five sows, found in unilateral side of the sperm reservoir in one sow and found in both sides of the sperm reservoir in one sow. No spermatozoa were found in the cranial isthmus, while only one spermatozoon was found in the ampulla part of a sow in the IUI group. In conclusion, DIUI resulted in a significantly lower number of spermatozoa in the sperm reservoir approximately 24 h after insemination compared with AI and IUI. Spermatozoa could be obtained from both sides of the sperm reservoir after AI and IUI but in one out of five sows inseminated by DIUI.     

Sperm Storage in the Female Reproductive Tract in Birds

The ability to store sperm in the female genital tract is frequently observed in vertebrates as well as in invertebrates. Because of the presence of a system that maintains the ejaculated sperm alive in the female reproductive tract in a variety of animals, this strategy appears to be advantageous for animal reproduction. Although the occurrence and physiological reasons for sperm storage have been reported extensively in many species, the mechanism of sperm storage in the female reproductive tract has been poorly understood until recently. In avian species, the specialized simple tubular invaginations referred to as sperm storage tubules (SSTs) are found in the oviduct as a sperm storage organ. In this review, we summarize the current understanding of the mechanism of sperm uptake into the SSTs, maintenance within it, and controlled release of the sperm from the SSTs. Since sperm storage in avian species occurs at high body temperatures (i.e., 41 C), elucidation of the mechanism for sperm storage may lead to the development of new strategies for sperm preservation at ambient temperatures, and these could be used in a myriad of applications in the field of reproduction.     

The bovine oviduct and its role in reproduction: a review of the literature

The bovine oviduct provides the environment for sperm transport and capacitation, oocyte transport and maturation, fertilization and early embryonic cleavage. Gamete interactions in the tube occur in contact with both the tubal epithelium and the oviduct fluid secreted by these cells. Current research continues to reveal the active role of the oviduct and its products play in normal fertilization and embryo development. This paper reviews the anatomy and physiology of the oviduct of the cow, including the specific events of reproduction which occur in this organ.     

Regulation of Capacitation of Canine Spermatozoa during Co-culture with Heterologous Oviductal Epithelial Cells

Progress of essential steps of the capacitation is coordinated in the oviductal isthmus, where sperm are stored in close contact with the epithelium. A crucial capacitational event is the phosphorylation of sperm membrane proteins. Regulation of the tyrosine phosphorylation by the oviduct has not been examined in dog sperm yet. The aim of this work was to study the effect of dog sperm binding to porcine oviductal epithelium on capacitation‐induced cellular and molecular changes. Epithelial cells were stripped from the oviducts of post‐puberal sows and cultured for 5–7 days at 39°C and 5% CO₂ on Biomatrix‐covered Chamber slides. Sperm washed through Percoll was co‐incubated with the oviductal epithelium cell cultures in a bicarbonate Tyrode's medium. During co‐incubation, sperm membrane changes, the state of tyrosine phosphorylation and motility were determined after 3, 30, 90, 180, 240 and 360 min. Significant increases in the percentage of capacitated and dead cells were observed in unbound sperm, while bound sperm remained uncapacitated, live and motile. An increasing tyrosine phosphorylation of tail proteins in bound, unbound and control sperm suspensions and a subsequent phosphorylation of head proteins in unbound and control sperm suspensions were observed. A significant difference regarding head phosphorylation (p < 0.05) was found between sperm bound to oviductal epithelium and unbound sperm. Binding occurred mainly in sperm with non‐ phosphorylated heads, while higher proportions of phosphorylated cells were found in unbound populations. The head phosphorylation progressed significantly during incubation in unbound spermatozoa (p < 0.05); however, it was suppressed in population of sperm attached to oviductal epithelium. Significant correlations between motility parameters related to hyperactivation and tail phosphorylation were found in unbound sperm. These observations support the hypothesis that spermatozoa with non‐phosphorylated heads preferentially attach to epithelial cells. It can be concluded that tyrosine phosphorylation of head membrane proteins and capacitation are delayed in canine spermatozoa being in closed contact with oviductal epithelium.     

Effect of Sperm Cryopreservation and Supplementing Semen Doses with Seminal Plasma on the Establishment of a Sperm Reservoir in Gilts

Frozen-thawed (FT) boar sperm have a reduced fertile life, due in part to a capacitation-like status induced by cooling. Reversal of this cryocapacitation in vitro by exposure to boar seminal plasma (SP) has been demonstrated. The objective of these studies was to determine the effect of SP on the ability of FT sperm to create an oviductal sperm reservoir following artificial insemination (AI). In Experiment one, 35 pre-pubertal gilts were injected (IM) with 400 IU eCG plus 200 IU hCG to induce oestrus. At detection of oestrus, gilts were inseminated with 3 x 10(9) live sperm, either fresh (FS; n = 13), FT (n = 10), or FT supplemented with 10% v/v SP (n = 12). Gilts were killed 8 h later, their reproductive tracts recovered and the uterotubal junctions (UTJs) flushed to recover sperm. Fewer (p < 0.01) sperm were recovered following FT, compared to FS, inseminations, and there was no evident effect of SP. In Experiment two, 30 pre-pubertal gilts received IM injections of 1000 IU eCG followed by 5 mg pLH 80 h later to control time of ovulation. Gilts were inseminated with 3 x 10(9) live FS sperm (n = 6), FT sperm (n = 15) or FT sperm plus 10% SP (n = 9) at 12 h before ovulation and then sacrificed 8 h later. The UTJs were dissected and flushed for sperm recovery. Fewest (p < 0.001) sperm were recovered following FT insemination and there was no evident effect of SP. These data demonstrate that the size of the sperm reservoir is markedly reduced in gilts inseminated with FT sperm. However, the lack of effect of SP suggested that either it did not reverse cryocapacitation or that such a reversal does not impact the in vivo ability to create a sperm reservoir.     

Distribution and viability of spermatozoa in the canine female genital tract during post-ovulatory oocyte maturation

Background

Unlike other domestic mammals, in which metaphase-II oocytes are ovulated, canine ovulation is characterized by the release of primary oocytes, which may take 12 to up to 36 hours. Further 60 hours are needed for maturation to secondary oocytes which then remain fertile for about 48 hours. Oestrus takes 7 to 10 days on average and may start as early as a week before ovulation. This together with the prolonged process of post-ovulatory oocyte maturation requires an according longevity of spermatozoa in the female genital tract in order to provide a population of fertile sperm when oocytes have matured to fertilizability. Therefore the distribution and viability of spermatozoa in the bitch genital tract was examined during post-ovulatory oocyte maturation.

Methods

Thirteen beagle bitches were inseminated on the day of sonographically verified ovulation with pooled semen of two beagle dogs containing one billion progressively motile spermatozoa. Ovariohysterectomy was performed two days later (group 1, n = 6) and four days later (group 2, n = 7). The oviduct and uterine horn of one side were flushed separately and the flushing’s were checked for the presence of gametes. The oviducts including the utero-tubal junction and the uterine horns, both the flushed and unflushed, were histologically examined for sperm distribution.

Results

The total number of spermatozoa recovered by flushing was low and evaluation of viability was limited. Prophase-I oocytes were collected from oviduct flushing in group 1, whereas unfertilized metaphase-II oocytes were detected in group 2. From day 2 to day 4 after ovulation a significant decrease in the percentage of glands containing sperm (P<0.05) and a marked reduction of the mean sperm number in uterine horn glands were observed. A concomitant diminution of spermatozoa was indicated in the utero-tubal junction accompanied by a slight increase in sperm numbers in the mid oviduct.

Conclusions

Oocyte maturation to metaphase-II stage is accompanied by a continuous sperm detachment and elimination in the uterine horns. Entrance of spermatozoa into the caudal oviduct seems to be steadily controlled by the utero-tubal junction thus providing a selected sperm population to be shifted towards the site of fertilization when oocyte maturation is completed.     

In Vivo and In Vitro Sperm Interaction with Oviductal Epithelial Cells of Llama

Sperm reservoirs in South American Camelids would be crucial for successful fertilization. Since ovulation occurs approximately 36 h after mating, the maintenance of the sperm viability in the oviduct waiting for the ovum is a critical reproductive event. Our study aimed at determining whether the isthmus or the utero tubal junction (UTJ) could function as a sperm reservoir in llama by means of in vivo and in vitro experiments. For the in vivo experiments, the oviducts of adult females with a dominant follicle bigger than 7 mm were examined for the presence of sperm at 6, 18, 24, 28 and 35 h after mating. The results using scanning and transmission electron microscopy showed ultrastructural differences between isthmus and UTJ with respect to (1) predominance of secretory cells in the UTJ and ciliated cells in the isthmus epithelium and (2) cytoplasmic bulbous projection of the secretory cells in the UTJ. Sperm adhered by a mucus‐like substance were seen only in the UTJ at 6, 18, 24 and 28 h post‐mating. Lack of sperm adhered to oviductal mucosa was observed around ovulation (35 h). In vitro experiments demonstrated higher ability of UTJ epithelial cell explants with respect to isthmus explants to bind sperm in a co‐cultured system. The anatomical features and the presence of a sperm bonding agent in the UTJ together with the in vitro differential binding of sperm to UTJ explants strongly suggest that both may be feasible mechanisms that facilitate sperm storage in this oviductal region in llama.