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.     

Implantation in both of the uterine horns after the unilateral intrauterine insemination in rabbits.

Artificial insemination was carried out by injecting semen into the upper part of the left uterine horn in rabbits with a duplex uterus. Ovulation was then induced by administration of hCG. As a result, implanted fetuses were observed not only in the left uterine horn but also in the right uterine horn. However, when similar insemination was carried out after ligation of the right utero-tubal junction, fertilization did not occur in the right oviduct. From these finding, we conclude that some of the spermatozoa injected into the left uterine horn was discharged into the vagina, entered the right uterine cervical canal, and reached the right oviduct to fertilize the ova.     

Lysophosphatidic Acid Synthesis and its Receptors’ Expression in the Bovine Oviduct During the Oestrous Cycle

Lysophosphatidic acid (LPA) is a naturally occurring simple phospholipid which in the bovine reproductive system can be produced in the endometrium, corpus luteum, ovarian follicle and embryo. In this study, we examined the possibility that LPA receptors are expressed, and LPA synthesized, in the bovine oviduct. We found that the concentration of LPA was highest in infundibulum in the follicular phase of the oestrous cycle and was relatively high during the early‐luteal phase in all examined parts of the oviduct. We also documented that LPA synthesis engages both available pathways for LPA production. The autotaxin (ATX) protein expression was significantly higher in the infundibulum compared to the isthmus during the follicular phase of the oestrous cycle. During the early‐luteal phase of the oestrous cycle, ATX and phospholipase A2 (PLA2) protein expression was highest in ampulla, although the expression of LPARs was not as dynamic as LPA concentration in the oviduct tissue, and we presume that in the bovine oviduct, the most abundantly expressed receptor is LPAR2. In conclusion, our results indicate that the bovine oviduct is a site of LPA synthesis and a target for LPA action in the bovine reproductive tract. We documented that LPAR2 is the most abundantly expressed in the bovine oviduct. We hypothesize that in the bovine oviduct, LPA may be involved in the transport of gametes, fertilization and cellular signalling between the oviduct and cumulus–oocyte complex.     

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.     

Role of the oviduct in early embryo development

This review highlights the role of the oviduct in early embryo development, which has to fulfil many aligned and well-tuned tasks during early embryogenesis. The oviductal lining is subjected to dynamic changes to timely accomplish gamete transport, fertilization and embryo development and to deliver a competent and healthy conceptus to the endometrium which can implant and develop to term. Although knowledge about the role of the oviduct is limited, we know that embryos are very sensitive to the environment in which they develop. The success of in vitro embryo production techniques demonstrates that it is possible to bypass the oviduct during early development and, to a certain extent, replicate the conditions in vitro. However, comparative studies show that embryos developed in vivo are superior to their in vitro produced counterparts, underlining our relatively poor knowledge of the biology of the oviduct. Oviduct activity is orchestrated by various factors, depending on cyclic dynamics, which crucially affect the success of tubal transfer and/or (re-)collection of embryos in embryo transfer studies. This paper reviews data which demonstrate that in vivo culture of embryos in the bovine oviduct is a useful tool for the assessment of embryos developed under various conditions (e.g. superovulation vs single ovulation, lactating dairy cows vs non-lactating cows). It is concluded that more work in the field of early embryo development within the oviduct would contribute to improved ART protocols leading to healthy pregnancies and offspring.     

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.     

Follicle culture enhances fertilizability and cleavage of bovine oocytes matured in vitro

The fertilization and cleavage of bovine oocytes matured by intra- or extra-follicular methods were investigated. Oocytes were fertilized in vitro or in the rabbit oviduct and cleavage was assessed after in vitro culture of in vitro fertilized oocytes and after in vivo culture (rabbit oviducts) of xenogenously fertilized oocytes. The effect of fertilization with fresh-diluted or frozen-thawed semen were also examined. The intra-follicular method did not increase the nuclear maturation rate as compared with the extra-follicular method (57.9 and 52.7%, respectively). However, the proportions of in vitro fertilized eggs (54.8%) and of cleaved eggs (two to eight cells; 34.6%) in the rabbit oviduct for 48 h after xenogenous fertilization were higher (P less than .025) in the intra-follicular oocytes than those of the extra-follicular oocytes (37.1 and 21.3%, respectively). It was also found that the use of fresh-diluted semen resulted in more cleaved eggs from the rabbit oviduct than the use of frozen-thawed semen (43.4 and 23.3% in the intra-follicular oocytes, P less than .025; 31.0 and 7.8% in the extra-follicular oocytes, P less than .05), while the appearance of cleaved eggs following in vitro fertilization was extremely low (0 to 6.6%). The present results demonstrated that the intra-follicular culture method of bovine oocytes provided a physiological environment for cytoplasmic maturation leading to higher fertilizability and development than the conventional in vitro culture of extra-follicular oocytes.     

The distribution and immunolocalization of fibroblast growth factors (FGFs) in the rat oviduct during early pregnancy and the post-partum period

The mammalian oviduct provides a favourable environment for several reproductive processes, including ovum transport, sperm capacitation, fertilization and pre-implantation embryonic development. This environment is regulated by cyclic ovarian steroids, that is oestrogen, and growth factors. Fibroblast growth factors (FGFs) regulate the differentiation and growth of various cell types in the female genital tract. This study aimed to determine the localization of FGF1, FGF2, FGF receptor 1 (FGFR1) and 2 (FGFR2) in the rat oviduct, by immunohistochemistry, on day 5 of pregnancy and post-partum days 1, 3 and 5, and to demonstrate the possible functions of these proteins during early pregnancy and the post-partum period. On all examination days, cytoplasmic and nuclear FGF1 immunoreactivity was detected in the epithelium lining the infundibulum, ampulla and isthmus of the oviduct. Immunoreactivity was much stronger in the basal bodies of the cilia on the epithelium lining the infundibulum and ampulla. FGF1 immunoreactivity was also detected in stromal cells, myocytes and endothelial cells. Cytoplasmic FGF2 immunoreactivity was observed in the tunica muscularis, vascular myocytes and endothelial cells. While strong cytoplasmic FGF2 immunoreactions were observed in the stromal cells of the lamina propria, the luminal epithelium, some stromal cells and smooth muscle cells displayed a rather weak FGFR1 and FGFR2 immunoreactivity. Immunoreaction intensity did not differ between the periods examined. This study shows that FGF1, FGF2, FGFR1 and FGFR2 are produced by rat oviduct cells during pregnancy and the post-partum period, and reproductive physiology is regulated not only by hormonal mechanisms, but also by growth factors.     

Formation of a Reservoir of Sperm in the Oviduct

A reservoir of sperm in the initial segment of the oviduct has been found in several species of domestic and laboratory mammals. Evidently, the reservoir serves to ensure successful fertilization by providing the appropriate number of sperm in the appropriate physiological state for fertilizing oocytes soon after they enter the oviduct. Recent evidence indicates that sperm are trapped in the reservoir by binding to specific carbohydrate moieties on the surface of the mucosal epithelium of the oviduct. A bovine seminal plasma protein has been identified that associates with sperm and confers on them the capacity to bind to the carbohydrate moiety.     

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.     

牦牛输卵管上皮细胞和卵泡颗粒细胞的培养

分离培养牦牛输卵管上皮细胞和卵泡颗粒细胞的目的是克服体外受精中早期胚胎发育阻断,建立有利于牦牛早期胚胎体外发育的共培养体系.采集牦牛输卵管上皮细胞进行原代及传代培养,同时从卵泡液中获取颗粒细胞进行原代培养,培养过程中观察2种细胞的生长方式和形态特点.输卵管上皮细胞贴壁生长时,呈多边形,且呈单层成簇生长.培养144 h～216 h,可形成细胞单层.颗粒细胞贴壁生长时,呈现聚集生长特性,贴壁细胞形状不规则,呈放射状.培养72 h～96 h,形成颗粒细胞单层.     

Effects of Retinoids on the In Vitro Development of Capra Hircus Embryos to Blastocysts in Two Different Culture Systems

The objective of this study was to evaluate the effect of retinol (RT) and retinoic acid (RA) on the in vitro development of pre‐implantation goat embryos cultured in potassium simplex optimized medium or synthetic oviduct fluid or cocultured in oviductal cells monolayer either in potassium simplex optimized medium or synthetic oviduct fluid. A total of 2407 cumulus‐oocyte complexes were aspirated from 2 to 6 mm ovarian follicles from slaughtered animals. Selected cumulus‐oocyte complexes were subjected to in vitro maturation in TCM 199 for 24 h at 39°C in an atmosphere of 5% (v/v) CO₂ in humidified air. In vitro fertilization was performed in modified defined medium. Eighteen hours after in vitro fertilization, cumulus cells were removed and presumptive zygotes were randomly distributed into experimental groups. In Experiment 1, presumptive zygotes were cultured in potassium simplex optimized medium, potassium simplex optimized medium + RT, potassium simplex optimized medium + retinoic acid, synthetic oviduct fluid, synthetic oviduct fluid + RT and synthetic oviduct fluid + RA at 39°C in a humidified atmosphere of 5% (v/v) CO₂, 5% (v/v) O₂ and 90% (v/v) N₂. In Experiment 2, presumptive zygotes were cocultured in potassium simplex optimized medium + oviductal cells monolayer, potassium simplex optimized medium + RT + oviductal cells monolayer, potassium simplex optimized medium + RA + oviductal cells monolayer, synthetic oviduct fluid + oviductal cells monolayer, synthetic oviduct fluid + RT + oviductal cells monolayer and synthetic oviduct fluid + RA + oviductal cells monolayer in an atmosphere of 5% (v/v) CO₂ in humidified air. In both experiments, media were partially changed on day 2 after in vitro fertilization and unfertilized oocytes were excluded from the experiment. Embryos were cultured or cocultured for 8 days. In Experiment 1, there was no effect of RT or RA supplementation on the proportion of oocytes that reached the morula or blastocyst stages. By contrast, Experiment 2 demonstrated that the addition of 0.28 μg/ml RT and 0.5 μm RA to the embryo culture media stimulated (p < 0.05) development to the morula and blastocyst stages under the coculture conditions tested. In conclusion, retinoids play an important role in pre‐implantation development of goat embryos and can be used to enhance in vitro embryo production.     

An Immunohistochemical Observations on the Oviduct of the Goat

The oviduct has an important role in regulating transport of gametes and fertilization. The main role in these functions has a smooth muscle cells and ciliated epithelium lining the oviduct. All functions are under the influence of hormonal and nervous system. The objective of this study was immunohistochemically to examine the following structures: lining epithelium, smooth muscle cells, elastic fibres and nerve fibres. For this purpose, the following antibodies were used: cytokeratin 18, S‐100 protein, acetylated α‐tubulin, smooth muscle actin, desmin and elastin. Ciliary and secretory cells of the lining epithelium were positive for cytokeratin 18 and S‐100 protein. Cilia and the basal body‐associated structures of ciliary cells were positive to acetylated α‐tubulin. Smooth muscle cells (SMC) in mucosa and of the muscular layer were positive for α‐smooth muscle actin (SMA) and desmin. High density of nerve fibres positively reacted to acetylated α‐tubulin and S100 protein was present in the mucosa, muscular layer and serosa. Elastic fibres positive for elastin form a dense network at the base of the mucosal folds and in the muscle layer. A dense network of these fibres is accompanying the blood vessels. It is supposed that together with smooth muscle cells they are involved in the transport of ovum and in blood flow regulation.     

The Influencing Factor of In Vitro Fertilization and Embryonic Transfer in the Domestic Fowl (Gallus domesticus)

This study was conducted to explore the influencing factors of ova in vitro fertilization (IVF) and transfer of the fertilized ova into the oviduct of recipient hens. The efficiency of fertilization was compared using three aspects: (i) the different time of ova collection and transfer, (ii) egg‐laying period of recipient hen; and (iii) semen volume. The following results are observed: 72%, 40% and 0% of ova were found in ovarian sac in 30～40 min, 50～60 min and more than 90 min post‐oviposition, respectively; 20%, 18%, 14% and 5.8% of ova were fertilized with 0.1, 0.2, 0.5 and 1.0 ml semen, respectively; and 33% and 100% of healthy chickens were hatched from fertile ova with 0.1 and 0.5 ml of semen, respectively. All oocytes obtained from ovary and mid‐oviduct were unfertilized. Embryos were transferred into recipient hens 30 min ± 10 min post‐oviposition, and 70% of shelled eggs were produced. There were no eggs produced in the other transfer times. This demonstrated that live chicken can be obtained by IVF of ova collected shortly after oviposition. It was important that the ovum was transferred into the oviduct infundibulum of recipient hens immediately or shortly after oviposition.     

Effect of hCG and eCG Treatments on Prostaglandins Synthesis in the Porcine Oviduct

The oviduct plays a crucial role in fertilization, gamete maturation and embryo transport. Prostaglandins are some of the main factors determining its roles. The present study investigated the influence of oestrus synchronization and superovulation on prostaglandins synthesis in the porcine oviduct. Mature cross‐bred gilts after exhibiting oestrous cycles were divided into four groups: I, cyclic; II, inseminated; III, synchronized and inseminated; and IV, superovulated and inseminated. Oviducts were collected on the third day of the oestrous cycle or after insemination and divided into isthmus and ampullary parts. This study demonstrated lower mRNA (in the isthmus and ampulla; p < 0.05, p < 0.001, respectively) and protein prostaglandin endoperoxide synthase 2 expression (in the isthmus; p < 0.001) in gilts treated with human chorionic gonadotrophin/equine chorionic gonadotrophin (hCG/eCG) compared with Group II that were inseminated only. In addition, hCG and eCG treatment decreased mPGES‐1 mRNA levels in Groups III and IV, in both the isthmus (p < 0.01 in III, p < 0.001 in IV) and ampulla (p < 0.001). The prostaglandin E₂ content of oviductal tissues was significantly lower in Groups III (p < 0.05) and IV (p < 0.01 in isthmus, p < 0.0001 in ampulla) compared with Group II. mRNA and protein levels of PGFS in Group IV in the oviductal isthmus were higher (p < 0.01) compared with the non‐treated Group II. In effect, the amount of prostaglandin F_2α in oviductal tissues of gilts treated with hCG/eCG was significantly elevated (p < 0.001 in isthmus of Groups III and IV; p < 0.05 in ampulla of Group IV). Differential expression of the factors analysed in gilts treated with exogenous gonadotrophins suggests that hCG/eCG stimulation affects prostaglandins synthesis pathway. These changes can alter oviduct functions and in turn affect gamete maturation and fertilization as well as development of embryos and their transport to the uterus.     

Cholinergic innervation of the oviduct in rabbits]

Distribution of cholinergic nerves in the oviduct wall in rabbits was investigated by a neurohistochemical method after El Badawi and Schenk (1967). Higher density of cholinergic nerve fibres was confirmed to occur mainly in the isthmic part, but in contrast with literary data it was also found in the ampullar part of oviducts. In the basal parts of oviduct mucosa there were only few cholinergic nerve fibres unlike their density in woman's oviduct. A major part of cholinergic nerves are concentrated in a thick muscular tissue; this is probable to be related with its influence on oviduct motility. In the course of pregnancy, progesterone reduces the number of cholinergic nerves in rabbit oviducts, the number is largely reduced in all layers. These results are considered as preliminary with respect to the number of test rabbits; they indicate that the rabbit oviduct is not any ideal model to study the development of cholinergic innervation in dependence on oestrous cycle phase and pregnancy duration and that they should not be applied to changes in the functional neuromorphology of tuba uterina in woman.     

N-acetylglucosaminyltransferase I activity of bovine oviduct epithelial cells: stimulation by luteinizing hormone, vascular endothelial growth factor and tumor necrosis factor alpha

N-acetylglucosaminyltransferase I (GnT I; EC 2.4.1.101), which catalyzes the first step in the conversion of oligomannose to complex or hybrid N-glycans of glycoproteins, was found in media cultured with bovine oviduct epithelial cells (BOEC) obtained from non-pregnant cows during the follicular phase. Combined treatment with specific hormones increased GnT I release from BOEC. Luteinizing hormone (LH; 10 ng/ml) alone slightly, but together with 17beta-estradiol (E2; 1 ng/ml), synergistically increased GnT I activity. Vascular endothelial growth factor (VEGF) and tumor necrosis factor (TNF) alpha, which have been shown to have their highest activities in the bovine oviduct during the periovulatory period, also increased in GnT I activity. This study provides the first evidence of an increase of GnT I release from BOEC in vitro, and shows that endocrine as well as local factors such as LH, VEGF and TNFalpha increase this activity. The results suggest that GnT I activity in the bovine oviduct may contribute to the induction of glycosylation and thereby contributing to the provision of the optimal microenvironment for fertilization and early development of the embryos.     

Heterogenous and xenogenous fertilization of in vivo matured equine oocytes

Forty-five in vivo matured equine oocytes were recovered from 63 follicular aspiration attempts (71.4%). HCG did not improve recovery rate (65% — 24/37 for treated vs 81% — 21/26 for nontreated mares). Fifteen oocytes were transferred into the oviduct of inseminated recipient mares (heterogenous fertilization) and 15 oocytes plus equine spermatozoa were transferred into rabbit oviducts (xenogenous fertilization). Ten oocytes (3 fertilized) were recovered from recipient mare oviducts following removal and flushing two days after transfer. Eight oocytes (nonfertilized) were recovered from rabbit oviducts. Oviductal transfer into separate recipient mares of three embryos produced from heterogenous fertilization resulted in two pregnancies. One mare produced a normal live foal and the other mare aborted at 20 days of gestation. Results from these studies suggest that: 1) a reliable method for collection of in vivo matured oocytes has been established, and 2) heterogenous fertilization is a technique that with refinement should be immediately applicable to obtain foals from valuable infertile mares that fail to get pregnant or produce embryos by standard methods.