Sperm functionality is differentially regulated by porcine oviductal extracellular vesicles from the distinct phases of the estrous cycle.

Context Extracellular vesicles (EVs) derived from the oviductal fluid (oEVs) play a critical role in various reproductive processes, including sperm capacitation, fertilisation, and early embryo development. Aims To characterise porcine oEVs (poEVs) from different stages of the estrous cycle (late follicular, LF; early luteal, EL; mid luteal, ML; late luteal, LL) and investigate their impact on sperm functionality. Methods poEVs were isolated, characterised, and labelled to assess their binding to boar spermatozoa. The effects of poEVs on sperm motility, viability, acrosomal status, protein kinase A phosphorylation (pPKAs), tyrosine phosphorylation (Tyr-P), and in in vitro fertility were analysed. Key results poEVs were observed as round or cup-shaped membrane-surrounded vesicles. Statistical analysis showed that poEVs did not significantly differ in size, quantity, or protein concentration among phases of the estrous cycle. However, LF poEVs demonstrated a higher affinity for binding to sperm. Treatment with EL, ML, and LL poEVs resulted in a decrease in sperm progressive motility and total motility. Moreover, pPKA levels were reduced in presence of LF, EL, and ML poEVs, while Tyr-P levels did not differ between groups. LF poEVs also reduced sperm penetration rate and the number of spermatozoa per penetrated oocyte (P Conclusions poEVs from different stages of the estrous cycle play a modulatory role in sperm functionality by interacting with spermatozoa, affecting motility and capacitation, and participating in sperm-oocyte interaction. Implications The differential effects of LF and LL poEVs suggest the potential use of poEVs as additives in IVF systems to regulate sperm-oocyte interaction.

Ovarian sex steroid and epithelial control of immune responses in the uterus and oviduct: human and animal models†.

The female reproductive tract (FRT), including the uterus and oviduct (Fallopian tube), is responsible for maintaining an optimal microenvironment for reproductive processes, such as gamete activation and transportation, sperm capacitation, fertilization, and early embryonic and fetal development. The mucosal surface of the FRT may be exposed to pathogens and sexually transmitted microorganisms due to the opening of the cervix during mating. Pathogens and endotoxins may also reach the oviduct through the peritoneal fluid. To maintain an optimum reproductive environment while recognizing and killing pathogenic bacterial and viral agents, the oviduct and uterus should be equipped with an efficient and rigorously controlled immune system. Ovarian sex steroids can affect epithelial cells and underlying stromal cells, which have been shown to mediate innate and adaptive immune responses. This, in turn, protects against potential infections while maintaining an optimal milieu for reproductive events, highlighting the homeostatic involvement of ovarian sex steroids and reproductive epithelial cells. This article will discuss how ovarian sex steroids affect the immune reactions elicited by the epithelial cells of the non-pregnant uterus and oviduct in the bovine, murine, and human species. Finally, we propose that there are regional and species-specific differences in the immune responses in FRT.

Sperm selection by the oviduct: perspectives for male fertility and assisted reproductive technologies†.

The contribution of sperm to embryogenesis is gaining attention with up to 50% of infertility cases being attributed to a paternal factor. The traditional methods used in assisted reproductive technologies for selecting and assessing sperm quality are mainly based on motility and viability parameters. However, other sperm characteristics, including deoxyribonucleic acid integrity, have major consequences for successful live birth. In natural reproduction, sperm navigate the male and female reproductive tract to reach and fertilize the egg. During transport, sperm encounter many obstacles that dramatically reduce the number arriving at the fertilization site. In humans, the number of sperm is reduced from tens of millions in the ejaculate to hundreds in the Fallopian tube (oviduct). Whether this sperm population has higher fertilization potential is not fully understood, but several studies in animals indicate that many defective sperm do not advance to the site of fertilization. Moreover, the oviduct plays a key role in fertility by modulating sperm transport, viability, and maturation, providing sperm that are ready to fertilize at the appropriate time. Here we present evidence of sperm selection by the oviduct with emphasis on the mechanisms of selection and the sperm characteristics selected. Considering the sperm parameters that are essential for healthy embryonic development, we discuss the use of novel in vitro sperm selection methods that mimic physiological conditions. We propose that insight gained from understanding how the oviduct selects sperm can be translated to assisted reproductive technologies to yield high fertilization, embryonic development, and pregnancy rates.

Dynamics of gametes and embryos in the oviduct: what can in vivo imaging reveal?

In brief: In vivo imaging of gametes and embryos in the oviduct enables new studies of the native processes that lead to fertilization and pregnancy. This review article discusses recent advancements in the in vivo imaging methods and insights which contribute to understanding the oviductal function. Abstract: Understanding the physiological dynamics of gametes and embryos in the fallopian tube (oviduct) has significant implications for managing reproductive disorders and improving assisted reproductive technologies. Recent advancements in imaging of the mouse oviduct in vivo uncovered fascinating dynamics of gametes and embryos in their native states. These new imaging approaches and observations are bringing exciting momentum to uncover the otherwise-hidden processes orchestrating fertilization and pregnancy. For mechanistic investigations, in vivo imaging in genetic mouse models enables dynamic phenotyping of gene functions in the reproductive process. Here, we review these imaging methods, discuss insights recently revealed by in vivo imaging, and comment on emerging directions, aiming to stimulate new in vivo studies of reproductive dynamics.

Morphogenesis of the uterine tube in the European shorthair cat in the prenatal and postnatal period: Light microscopy and three-dimensional reconstruction study.

The study aimed to describe the morphogenesis of the domestic cat uterine tube, from the primordial tubal segment of the paramesonephric duct (TsPD) to the mature organ. The specific aims included identifying differences in the rate and way of development of the layers in the wall of the infundibulum, the ampulla, and the isthmus. In the study we conducted light microscopic (LM) observations of the uterine tube wall, and analysis of 3D models, which were used for the first time to visualize the arrangement of the paramesonephric duct and highlight the pattern of the developing mucosal folds. The results revealed the two stages in the prenatal development of the domestic cat uterine tube. The first stage lasts from day 26-44 p.c. (post-conception) and includes the growth of the TsPD. It starts with formation of the TsPD's epithelial tubule in the mesenchyme of the anterior part of the mesonephros. The tubule, surrounded by mesenchyme, elongates posteriorly, and the TsPD can be divided into three sections according to their location and diameter, i.e., anterior, middle, and posterior. The first stage ends with the histodifferentiation of the mesenchyme into loose connective tissue and mucosa formation. The second stage lasts from approximately day 45-63 p.c. and comprises the formation of the proper layers in the uterine tube wall. The pace and pattern of layers' development are specific to each section of the uterine tube, as tissues first differentiate in its anterior part and then continue posteriorly. The mucosal folds appear after day 44 p.c. in the infundibulum, and they become long and branched by the end of the prenatal period. Then, approximately day 48 p.c., the single mucosal folds appear in the ampulla, and at the end, i.e., about day 63 p.c., they become visible in the isthmus. The 3D models revealed the unique spiral-shaped arrangement of the mucosal folds in the ampulla. The tunica muscularis starts to develop ca day 48-51 p.c. in the anterior part of the uterine tube. On approximately day 55 p.c., the circular muscle layer can be distinguished in the ampulla, while in the isthmus, it develops just before birth. The postnatal development of the infundibulum includes the formation of the labyrinth-like system of mucosal folds, whereas in the ampulla and isthmus, branched mucosal folds and the second longitudinal muscle layer develop postnatally. At the end of the prenatal period, the differences in the wall thickness between the individual segments of the uterine tube reflect the proper proportions of the uterine tube wall in the mature female.

Sperm-oviduct interaction: Differential gene expression of growth factors induced by sperm DNA fragmentation.

The present study investigated the effects of DNA fragmentation of spermatozoa on the growth factors expression by a human oviduct epithelial cell line (OE-E6/E7). Two separate groups were examined in this study. The cell line was cultured in the presence of spermatozoa with normal DNA fragmentation index (DFI) or abnormal DFI. Total RNA from the cell line in each group was isolated, and relative expression of objective genes was analysed using PCR array. Also, the concentration of VEGF, BMP-2, BMP-7 and MSTN in the supernatant of cell culture was analysed by the ELISA method. The PCR array analysis revealed that most of the growth factors had been upregulated in the abnormal group. However, the differences between groups were statistically significant (p < 0.05) for five genes, including VEGF-A, BMP-2, BMP-6, BMP-7 and OSM. Furthermore, MSTN was the only gene that down-regulated significantly under the influence of the spermatozoa with abnormal DFI. Moreover, the results of ELISA analysis were in agreement with the data of the PCR array. It has been concluded that DNA fragmentation in human spermatozoa can probably change regular events throughout the oviducts. Consequently, the genes of interest may change sperm function and probably its fate in the female reproductive tract.

From a Passive Conduit to Highly Dynamic Organ. What are the Roles of Uterine Tube Epithelium in Reproduction?

It is well known that the mammalian uterine tube (UT) plays a crucial role in female fertility, where the most important events leading to successful fertilization and pre-implantation embryo development occur. The known functions of these small intra-abdominal organs are: an uptake and transport of oocytes; storage, transportation, and capacitation of spermatozoa, and finally fertilization and transport of the fertilized ovum and early embryo through the isthmus towards the uterotubal junction. The success of all these events depends on the interaction between the uterine tube epithelium (UTE) and gametes/embryo. Besides that, contemporary research revealed that the tubal epithelium provides essential nutritional support and the most suitable environment for early embryo development. Moreover, recent discoveries in molecular biology help understand the role of the epithelium at the cellular and molecular levels, highlighting the factors involved in regulating the UT signaling, that affects different steps in the fertilization process. According to the latest research, the extracellular vesicles, as a major component of tubal secretion, mediate the interaction between gametes/embryo and epithelium. This review aims to provide up-to-date knowledge on various aspects concerning tubal epithelium activity and its cross-talk with spermatozoa, oocytes and preimplantation embryo and how these interactions affect fertilization and early embryo development.

Immunohistochemical and Scanning Electron Microscopic Confirmation of the Lymphatic Lacunae in the Uterine Tube Mucosal Folds. What Are the Clinical Implications?

Uterine tubes (UTs) are essential during physiological reproduction. The most intriguing part of its wall is the mucosa. Apart from the epithelial cells vital for its normal function, the connective tissue lamina propria contains wide spaces whose function, morphology and structure are yet to be elucidated. The present study used bioptic samples from 25 premenopausal (mean age 48,33 years, ?=3,56) and 25 postmenopausal women (mean age 57,8 years, ?=7,79). In both study groups, samples were obtained from two anatomically distinct parts of the UT - ampulla and infundibulum with fimbriae. The specimens were processed for scanning electron microscopy (SEM) and immunohistochemical detection of podoplanin (clone D2-40) and VEGFR-3 - two markers of lymphatic endothelial cells. The results showed that specimens from premenopausal and postmenopausal women contain wide lymphatic spaces, also known as lymphatic lacunae. The most probable function of the lacunae in the fimbriae is oocyte pick-up upon ovulation thanks to their ability to get engorged with lymph, thus serving as an erectile-like tissue. The ampullary lacunae are probably responsible for tubal fluid maintenance and recirculation. These results indicate that they are vital for normal reproduction because tubal fluid dynamics are as important as fluid composition. Further research on this topic is highly warranted because more detailed insights into UT function have a great potential to refine the methods of reproductive medicine, e.g. in vitro fertilization (IVF), which are still far from optimal regarding fertility outcomes.

Scanning electron microscopic study of the human uterine tube epithelial lining: surgical biopsy samples and epithelial cell culture.

This article summarizes the importance of the exact morphology of human uterine/fallopian tube epithelium at the scanning electron microscopy (SEM) level for the clinical outcome even nowadays. Visual referential micrographs from SEM reflect two ways to view human epithelial cell lining surfaces: the surface epithelial uterine tube from surgical tissue biopsy and human fallopian tube epithelial cells (HFTEC) culture monolayer surface. One colorized image visualizes ciliated cells, distinguishes them from non-ciliated cells, and provides an educational benefit. A detailed description of the ultrastructure in referential and pathologic human uterine tube epithelium is important in defining the morphological basis of high-grade carcinomas, in the mechanism of pathophysiology, and in discussing options for its prevention. Cell cultures of human fallopian tube epithelial cells offer new approaches in simulating the mechanisms of cancer genesis or may help to elucidate the genetic basis of several diagnoses. New technical approaches in SEM provide higher resolution and detailed surface images. The SEM modality is still one of the current options in diagnostics and may be useful for advancing human reproductive organ cancer research.

Cyclic activity of signal transduction pathways in fimbrial epithelium of the human fallopian tube.

INTRODUCTION: The local environment of the fallopian tube represents the optimal conditions for reproductive processes. To maintain tissue homeostasis, signal transduction pathways are thought to play a pivotal role. Enhancing our understanding of functional signal transduction pathway activity is important to be able to clarify the role of aberrant signal transduction pathway activity leading to female subfertility and other tubal diseases. Therefore, in this study we investigate the influence of the hormonal cycle on the activity of key signal transduction pathways in the fimbrial epithelium of morphologically normal fallopian tubes. MATERIAL AND METHODS: We included healthy pre- (n = 17) and postmenopausal (n = 8) patients who had surgical interventions for benign gynecologic conditions. Histologic sections of the fallopian tubes were reviewed by two pathologists and, for the premenopausal patients, hormone serum levels and sections of the endometrium were examined to determine the hormonal phase (early follicular [n = 4], late follicular [n = 3], early luteal [n = 5], late luteal [n = 5]). After laser capture microdissection, total mRNA was extracted from the fimbrial epithelium and real-time quantitative reverse transcription-PCR was performed to determine functional signal transduction pathway activity of the androgen receptor (AR), estrogen receptor (ER), phosphoinositide-3-kinase (PI3K), Hedgehog (HH), transforming growth factor-beta (TGF-ß) and canonical wingless-type MMTV integration site (Wnt) pathways. RESULTS: The early luteal phase demonstrated high AR and ER pathway activity in comparison with the late luteal phase (p = 0.016 and p = 0.032, respectively) and low PI3K activity compared with the late follicular phase (p = 0.036), whereas the late luteal phase showed low activity of HH and Wnt compared with the early follicular phase (both p = 0.016). Signal transduction pathway activity in fimbrial epithelium from postmenopausal patients was most similar to the early follicular and/or late luteal phase with regard to the AR, ER and PI3K pathways. Wnt pathway activity in postmenopausal patients was comparable to the late follicular and early luteal phase. We observed no differences in HH and TGF-ß pathway activity between pre- and postmenopausal samples. The cyclic changes in signal transduction pathway activity suggest a stage-specific function which may affect the morphology and physiology of the human fallopian tube. CONCLUSIONS: We demonstrated cyclic changes in activity of the AR, ER, PI3K, HH and Wnt pathways throughout the hormonal cycle.

Sperm migration in the genital tract-In silico experiments identify key factors for reproductive success.

Sperm migration in the female genital tract controls sperm selection and, therefore, reproductive success as male gametes are conditioned for fertilization while their number is dramatically reduced. Mechanisms underlying sperm migration are mostly unknown, since in vivo investigations are mostly unfeasible for ethical or practical reasons. By presenting a spatio-temporal model of the mammalian female genital tract combined with agent-based description of sperm motion and interaction as well as parameterizing it with bovine data, we offer an alternative possibility for studying sperm migration in silico. The model incorporates genital tract geometry as well as biophysical principles of sperm motion observed in vitro such as positive rheotaxis and thigmotaxis. This model for sperm migration from vagina to oviducts was successfully tested against in vivo data from literature. We found that physical sperm characteristics such as velocity and directional stability as well as sperm-fluid interactions and wall alignment are critical for success, i.e. sperms reaching the oviducts. Therefore, we propose that these identified sperm parameters should be considered in detail for conditioning sperm in artificial selection procedures since the natural processes are normally bypassed in reproductive in vitro technologies. The tremendous impact of mucus flow to support sperm accumulation in the oviduct highlights the importance of a species-specific optimum time window for artificial insemination regarding ovulation. Predictions from our extendable in silico experimental system will improve assisted reproduction in humans, endangered species, and livestock.

Metabolomic analysis of oviduct fluid on day 3 post-estrus in Holstein heifers.

The metabolites in the oviduct fluid (OF) of both oviducts were analyzed by proton nuclear magnetic resonance (1H-NMR) in Holstein heifers on day 3 after synchronized estrus. Twenty-six metabolites were quantified, among which lactate, glycine and myoinositol were the most abundant. Six metabolites including glycine and myoinositol varied in amount according to the proximity to the corpus luteum. Glucose and histidine were among the most variable metabolites among heifers while threonine and lactate were among the most stable ones, suggesting different mechanisms of homeostasis in the OF.

Advancements in Microfluidic Systems for the Study of Female Reproductive Biology.

The female reproductive tract is a highly complex physiological system that consists of the ovaries, fallopian tubes, uterus, cervix, and vagina. An enhanced understanding of the molecular, cellular, and genetic mechanisms of the tract will allow for the development of more effective assisted reproductive technologies, therapeutics, and screening strategies for female specific disorders. Traditional 2-dimensional and 3-dimensional static culture systems may not always reflect the cellular and physical contexts or physicochemical microenvironment necessary to understand the dynamic exchange that is crucial for the functioning of the reproductive system. Microfluidic systems present a unique opportunity to study the female reproductive tract, as these systems recapitulate the multicellular architecture, contacts between different tissues, and microenvironmental cues that largely influence cell structure, function, behavior, and growth. This review discusses examples, challenges, and benefits of using microfluidic systems to model ovaries, fallopian tubes, endometrium, and placenta. Additionally, this review also briefly discusses the use of these systems in studying the effects of endocrine disrupting chemicals and diseases such as ovarian cancer, preeclampsia, and polycystic ovarian syndrome.

Is It Possible to Treat Infertility with Stem Cells?

Infertility is a major health problem, and despite improved treatments over the years, there are still some conditions that cannot be treated successfully using a conventional approach. Therefore, new options are being considered and one of them is cell therapy using stem cells. Stem cell treatments for infertility can be divided into two major groups, the first one being direct transplantation of stem cells or their paracrine factors into reproductive organs and the second one being in vitro differentiation into germ cells or gametes. In animal models, all of these approaches were able to improve the reproductive potential of tested animals, although in humans there is still too little evidence to suggest successful use. The reasons for lack of evidence are unavailability of proper material, the complexity of explored biological processes, and ethical considerations. Despite all of the above-mentioned hurdles, researchers were able to show that in women, it seems to be possible to improve some conditions, but in men, no similar clinically important improvement was achieved. To conclude, the data presented in this review suggest that the treatment of infertility with stem cells seems plausible, because some types of treatments have already been tested in humans, achieving live births, while others show great potential only in animal studies, for now.

Cell-type specific analysis of physiological action of estrogen in mouse oviducts.

One of the endogenous estrogens, 17ß-estradiol (E2 ) is a female steroid hormone secreted from the ovary. It is well established that E2 causes biochemical and histological changes in the uterus. However, it is not completely understood how E2 regulates the oviductal environment in vivo. In this study, we assessed the effect of E2 on each oviductal cell type, using an ovariectomized-hormone-replacement mouse model, single-cell RNA-sequencing (scRNA-seq), in situ hybridization, and cell-type-specific deletion in mice. We found that each cell type in the oviduct responded to E2 distinctively, especially ciliated and secretory epithelial cells. The treatment of exogenous E2 did not drastically alter the transcriptomic profile from that of endogenous E2 produced during estrus. Moreover, we have identified and validated genes of interest in our datasets that may be used as cell- and region-specific markers in the oviduct. Insulin-like growth factor 1 (Igf1) was characterized as an E2 -target gene in the mouse oviduct and was also expressed in human fallopian tubes. Deletion of Igf1 in progesterone receptor (Pgr)-expressing cells resulted in female subfertility, partially due to an embryo developmental defect and embryo retention within the oviduct. In summary, we have shown that oviductal cell types, including epithelial, stromal, and muscle cells, are differentially regulated by E2 and support gene expression changes, such as growth factors that are required for normal embryo development and transport in mouse models. Furthermore, we have identified cell-specific and region-specific gene markers for targeted studies and functional analysis in vivo.

Biophysical optimization of preimplantation embryo culture: what mechanics can offer ART.

Owing to the rise of ART and mounting reports of epigenetic modification associated with them, an understanding of optimal embryo culture conditions and reliable indicators of embryo quality are highly sought after. There is a growing body of evidence that mechanical biomarkers can rival embryo morphology as an early indicator of developmental potential and that biomimetic mechanical cues can promote healthy development in preimplantation embryos. This review will summarize studies that investigate the role of mechanics as both indicators and promoters of mammalian preimplantation embryo development and evaluate their potential for improving future embryo culture systems.

Fluid deficit calculation at hysteroscopy: could consideration of intraperitoneal fluid accumulation add insight to safety limits?

BACKGROUND: The current literature and guidelines are largely silent regarding the contribution of the fallopian tubes to the fluid deficit (FD) during hysteroscopy. We explored whether the FD could be in part due to transtubal passage. METHODS: This was a prospective cohort study. Patients who underwent hysteroscopy because of benign gynecologic pathology with, or without, laparoscopy were enrolled. The fluid deficit and, in laparoscopic cases, the amount of fluid found in the pelvis were prospectively reported. RESULTS: Comparisons between FD and intraperitoneal fluid were performed. Sixty-five patients were included in the study. Forty-five underwent hysteroscopy prior to laparoscopy and 20 patients underwent hysteroscopy-only. These were further divided into operative hysteroscopy and diagnostic hysteroscopy subgroups. In the laparoscopy group, the average FD was 525.9 mL (95% CI: 482.1-569.7) and the calculated FD due to intravasation was 286.6 mL (95%CI: 253.0-320.3). In the hysteroscopy without laparoscopy group, the average FD was 303.0 mL (95% CI: 85.2-520.8). There was no correlation between the intrauterine fluid pressure and the amount of FD, or the presence of intraperitoneal fluid. CONCLUSIONS: Most women with patent tubes undergoing hysteroscopy have accumulation of distention fluid in the pelvis and that the passage was not correlated with the intrauterine fluid pressure. These findings add new insight to the current guidelines, suggesting more accurate and patient-centered safety protocols.

Early embryo-maternal communication in the oviduct: A review.

An intact embryo-maternal communication is critical for the establishment of a successful pregnancy. To date, a huge number of studies have been performed describing the complex process of embryo-maternal signaling within the uterus. However, recent studies indicate that the early embryo communicates with the oviductal cells shortly after fertilizationand that this is important for the successful establishment of pregnancy. Only if the early embryo is capable to signal the mother within a precise timeframe and to garner a response, will the embryo be able to survive and reach the uterus. This review will give an overview of all the experimental designs which have investigated embryo-maternal interaction in the oviduct. In addition to that, it will provide a comprehensive analysis of the findings to date elucidating the morphological and molecular changes in the oviduct which are induced by the presence of the early embryo highlighting how the tubal responses affect embryo development and survival.

Involvement of activin signal pathway in cyclic apoptosis of the oviductal isthmic epithelium in cows.

Activin (ACV) A induces various cellular functions via activin receptor type 2 (ACVR2A/2B)-activin receptor-like kinase (ALK) 4 -Smad 2/3 pathway. Although the production of ACVA is indicated in bovine oviducts, its role on the oviduct is unclear. Oviductal isthmus needs to change its function rapidly at peri-fertilization, however, the mechanism is unknown. This study was aimed to clarify the role of ACVA in the morphological changes of oviductal isthmus in cows. First, mRNA expressions of INHBA (ACVA component) and its receptors (ALK4, ACVR2A and ACVR2B) in the isthmic tissues were examined throughout the estrous cycle. INHBA was the highest, however, ACVR2A was the lowest on the day of ovulation, suggesting reduced ACV signal transduction in the isthmus just after ovulation. Proteins of ACVRs and Smad2/3 were clearly detected in the cultured epithelial cells. It is known that ACVA regulates cellular apoptosis. Our data showed that the number of cleaved caspase-3-positive epithelial cells was largest at 2-3 days after ovulation in the isthmus. Interestingly, our study demonstrated that follistatin (ACV/TGFB/BMP inhibitor) significantly decreased the BCL2/BAX ratio in the cultured isthmic epithelial cells. To clarify which ALK pathway is involved in the regulation of BCL2/BAX ratio, the effects of SB431542 (ACV signaling (ALK4) and TGFB signaling (ALK5) inhibitor), SB525334 (ALK5 inhibitor) and LDN193189 (BMP signaling (ALK2/3) inhibitor) were investigated in the next study. The results showed that only SB431542 significantly decreased BCL2/BAX and the others had no effects. These results suggest that decreased ACVA-ACVR2A-ALK4 signal at the post-ovulation induces cyclic apoptosis of isthmic epithelial cells in bovine oviducts.

Evolutionary sequences in mammalian reproductive biology.

Broad biological aspects and accepted evolutionary sequences may offer useful guidance towards a comprehensive explanation of the function of mammalian ovaries and oviducts and their vital contribution to the events of fertilization. Cooling of the preovulatory follicles before ovulation may well have its roots in the primitive stages of external fertilization in which aggregates of oocytes are shed into freshwater.