From sperm to embryos; lessons learnt from Tim Rowson's career.

L.E.A.Rowson (1914-1989) raised horizons in farm animal research and the impact of his reproductive contributions remains of major importance worldwide. First, his studies with E.J.C. Polge on the successful deep - freezing and thawing of bull spermatozoa enabled artificial insemination to be applied on an international scale. Second, his development of non-surgical embryo transfer techniques in cattle gave a major boost to the breeding industry, facilitated by deep -frozen storage of embryos during international transport. He also guided many research students whose work in cattle, sheep and horses has become prominent during the past 60 years.

Local cooling of the ovary and its implications for heat stress effects on reproduction.

The effects of season on the fertility of the dairy cow added to the metabolic stress of milk production are well known. We here present lactating dairy cows as a comparative model of this problem. This review examines the results of recent studies that have highlighted heat stress (HS) effects on pre-ovulatory follicles. From these studies, we draw information regarding the mechanisms giving rise to temperature gradients across reproductive tissues. Our review is completed by a discussion of approaches designed to reduce the negative effects of HS based on cooling strategies implemented before ovulation at or around estrus.

The presence of two ovulatory follicles at timed artificial insemination influences the ovulatory response to GnRH in high-producing dairy cows.

This study sought to examine the impact of the presence of two co-dominant (ovulatory) follicles at the time of artificial insemination (AI) on the ovulatory response to GnRH given in a fixed-time AI protocol. The study population comprised 622 lactating dairy cows: 306 (49.2%) with a single follicle, 198 (31.8%) with two bilateral follicles (one follicle per ovary) and 118 (19%) with two unilateral follicles (same ovary). Based on odds ratios, cows with two bilateral or unilateral follicles were less likely (by factors of 0.09 and 0.11, respectively) to undergo ovulation failure compared with cows with one follicle (P = 0.01 and P = 0.02, respectively); the likelihood of ovulation failure decreased 0.75 times with every one-mm increase in follicle diameter for cows with a single follicle, whereas individual follicle diameter was not related to ovulation failure in cows with two bilateral follicles (P = 0.001). The likelihood of double ovulation decreased 0.7 times with every one-mm diameter difference between the larger and smaller follicle for all cows with two follicles (P = 0.001), whereas cows with two unilateral follicles showed a higher (P < 0.05) double ovulation rate than cows with two bilateral follicles. In 116 (58.6%) of the cows with two bilateral follicles, only the larger follicle ovulated in 59.5% cows, whereas only the smaller one ovulated in the remaining 40.5% cows. In these cows, a one-mm size difference between the larger and the smaller follicle gave rise to a 1.12-fold increase in the ovulation failure rate for the larger follicles (P = 0.0001). Cows with two bilateral follicles were more likely (by a factor of 1.5) to conceive than cows with one follicle (P = 0.001). Significant right-left differences were not found in cows with two bilateral follicles, whereas the right ovary was more active than the left in the remaining cows. Our results indicate that cows with two co-dominant follicles at AI show different ovulation patterns to those with one dominant follicle. A higher rate of ovulation failure was observed among cows with one follicle than cows with two follicles, whereas the conception rate was higher for cows with two bilateral follicles than for the remaining cows. In cows with two follicles, double ovulations along with ovulation of the smaller follicle were related to the least size difference between the larger and smaller follicle.

From pre-ovulatory follicle palpation to the challenge of twin pregnancies: Clinical reflections following one million gynaecological examinations in dairy cows.

CONTENTS: Based on experimental studies and extensive field-scale experience, this review offers new proposals for: (i) elevating the success of modern insemination procedures, (ii) emphasizing features of a pre-ovulatory follicle in the context of optimum insemination timing, (iii) overcoming heat stress and its consequences using physiological processes or endocrine protocols, (iv) establishing a viable pregnancy or early pregnancy loss and (v) the challenge of twin pregnancies. In conclusion, the fertility of high-yielding dairy cows can be further improved.

Temperature gradients in vivo influence maturing male and female gametes in mammals: evidence from the cow.

Since 1980 several reports have indicated that temperatures vary between preovulatory follicles and other ovarian tissues in rabbit, cow, pig and human. However, these observations did not achieve prominence; they were regarded as artefacts due to the use of anaesthetics and open surgery (laparotomy). Recently, without resorting to anaesthesia or surgery, direct measurements of temperature in preovulatory follicles have been performed in the cow by means of a thermistor probe introduced into the antrum under ultrasonic guidance. Such follicles revealed a mean antral (follicular fluid) temperature 0.74°C and 1.54°C cooler than uterine surface and rectal temperatures respectively in ovulating cows, whereas no such temperature differences were detected in non-ovulating cows. Cows are predominantly monovular and preovulatory follicles attain a diameter of 15-22mm or more. These features and the timescale of response to the preovulatory gonadotrophin surge make them a valuable model for the human preovulatory follicle. Temperature gradients are interpreted primarily in a context of final maturation of gametes immediately before the onset of fertilisation. Preovulatory follicular temperature in women could be assessed by a comparable approach and might become a valuable selection guide for oocyte viability.

Charles Georges Thibault (1919-2003): inspirational leader in French reproductive biology.

The contribution of Charles Thibault in creating and developing a major international centre of animal research at Jouy-en-Josas (near Versailles) in post Second World War France is recorded in detail. Not only did he select a team of gifted young chercheurs, but he stimulated and supported their research in diverse ways. The projects covered were not only primarily of significance to animal reproduction, but they also became relevant to human infertility studies and to IVF treatments. Members of the team in Physiologie Animale gained international research reputations and seasoned overseas researchers were attracted to Jouy-en-Josas for their sabbaticals. Thibault himself was known especially for his studies on the mammalian oocyte, on fertilization both in vivo and in vitro, and for a key publication on parthenogenesis. Over and above leading the department of Physiologie Animale at the Institut National de la Recherche Agronomique (INRA), he was Professor of Reproductive Physiology at the University of Paris VI and an influential member of national committees in Paris. His formal career reached a peak with his appointment as President of the French scientific research organisation (CNRS), and his overall contributions were celebrated both nationally and internationally, not least as Commander of the Legion d'Honneur and recipient of the Wolf Prize, the latter being presented in the Israeli Parliament, the Knessett. His influence continues to be strong, felt in France and beyond through his protégés and his publications.

Cross-talk between free and bound spermatozoa to modulate initial sperm:egg ratios at the site of fertilization in the mammalian oviduct.

This essay proposes that highly localized communication between free and bound spermatozoa in the caudal portion of the oviduct acts to regulate the numbers detaching from the epithelium and progressing to the site of fertilization close to the time of ovulation. Low initial sperm:egg ratios are essential for monospermic fertilization. Liberation of surface macromolecules and metabolic prompting from activated spermatozoa, together with altered patterns of sperm movement and dynamic differences in intracellular Ca(2+) ion status between neighboring sperm cells, would influence the progressive release of spermatozoa from the reservoir in the oviduct isthmus. Different intensities of preovulatory epithelial binding, reflecting a range of states in the sperm surface membranes and associated proteins, would provide a further explanation for a chronologically staggered periovulatory detachment of spermatozoa. Intimate sperm-sperm interactions within the confines of the oviduct isthmus offer a sensitive means of fine-tuning the vanguard of competent male gametes reaching the isthmo-ampullary junction.

MC Chang--Reproductive biologist of distinction 1908-1991.

This article reviews the remarkable life and major scientific achievements of the reproductive biologist M.C. Chang. His scholarly career progressed from university in Peking, via Edinburgh, Scotland, and Cambridge, England, to the newly founded Worcester Foundation for Experimental Biology in Massachusetts. At each stage, the hand of fate is noted as are the support and encouragement of key professors. Chang's own contributions on capacitation of spermatozoa, in vitro fertilisation of mammalian eggs, and transplantation of oocytes and embryos are all brought out, as is his essential input to the creation and development of a steroid contraceptive pill. He strongly encouraged young reproductive biologists who worked in his laboratory, and applauded the world-wide distinction of his student and associate, R. Yanagimachi, as a specialist in mammalian fertilisation. Finally, Chang's continued feelings towards his homeland are contrasted with the reality of his American life after 1945, itself a study in poignancy.

Temperature gradients in female reproductive tissues.

Deep body temperature in mammals is generally but incorrectly regarded as uniform. Alterations of temperature in oviducts and preovulatory Graafian follicles may play a vital role in gamete maturation, fertilization and early embryonic development. At a molecular level, the conformation of regulatory proteins is susceptible to changes in temperature. Deviation from physiological temperature during IVF procedures could thereby exert a profound influence on patterns of gene expression as the embryonic genome unfolds during early cleavage stages and act to generate specific anomalies. Systematic studies are urgently required. The temperature of internal body organs in mammals such as rabbits and humans is widely regarded as uniform, but this is not correct. Temperatures in reproductive tissues such as ovaries and oviducts vary according to the stage of a menstrual or oestrous cycle. Such changes in temperature are thought to be critically involved in the maturation of eggs and spermatozoa and thus in events shortly before and after fertilization. Proteins in the cytoplasm and nuclei of eggs and very young embryos respond to small shifts in temperature by changing their three-dimensional shape. Conformational modifications in regulatory proteins in the nucleus would influence patterns of gene expression in developing embryos and may, when perturbed, alter the sexual phenotype of an individual. In the practical context of IVF, studies are needed of the influence of culturing gametes and embryos at different temperatures and ranges of temperature on the patterns of gene expression in preimplantation embryos.

Components of oviduct physiology in eutherian mammals.

Recalling the evolutionary sequence of development first of gonad and subsequently of oviducts, ovarian endocrine regulation of all known components of oviduct physiology is reviewed. Ovaries not only influence oviducts via the systemic blood circulation, but also locally by counter-current transfer of relatively high concentrations of steroid hormones and prostaglandins between the ovarian vein and oviduct branch of the ovarian artery. The efficiency and impact of such counter-current transfer is greatest around the time of ovulation, the transfer process receiving further inputs from hormones present in peritoneal fluid. Classical oviduct physiology is summarised, and the potential molecular consequences of temperature gradients within the duct lumen examined. At ovulation, an oocyte-cumulus complex is displaced in minutes from the follicular surface to the site of fertilisation at the ampullary-isthmic junction of the oviduct. This rapid initial phase is contrasted with the subsequent slow progression of embryos to the uterus in days, still encompassed within a zona pellucida. Regarding transport of spermatozoa, the formation of a pre-ovulatory reservoir in the caudal portion of the oviduct isthmus is noted, with suppression of motility and sperm-head binding to epithelial organelles acting to maintain fertilising ability. Completion of capacitation is prompted shortly before ovulation, predominantly by Ca(2+) influx into bound spermatozoa. A controlled release of spermatozoa coupled with their hyperactivation results in initial sperm:egg ratios at the site of fertilisation close to unity, thereby avoiding the pathological condition of polyspermy. Both the oviduct milieu and embryonic development are influenced by paracrine activity of follicular granulosa cells released at ovulation and remaining in suspension in the vicinity of the oocyte or embryo. These cells may amplify early pregnancy signals from a zygote to the endosalpinx. Beneficial effects of the oviduct on domestic animal embryos are contrasted with anomalies arising as a consequence of in vitro culture. Primate embryos do not require exposure to an oviduct for normal development, perhaps due to overlapping compositions of endosalpingeal and endometrial secretions. Additionally, primate endometrial secretions may be modified by viable gametes or an embryo in the presence of a cumulus cell suspension.

Intrafollicular insemination for the treatment of infertility in the dairy cow.

Intrafollicular insemination (IFI) is based on direct introduction of a sperm suspension into a preovulatory follicle. To our knowledge, the first cases of IFI resulting in pregnancy of the cow are reported here. The experiment was performed on a dairy herd with low fertility during the warm season of the year. Following the sequence of estrus detection, one in three inseminations was intrafollicular (n = 17), whereas deposition of semen was performed into the uterine body (IUI) in the remaining cows (n = 33). Approximately 0.06 ml of a seminal dose containing five million spermatozoa (one quarter of a commercial seminal dose in a 0.25 ml French straw) was injected trans-vaginally into the preovulatory follicle in the IFI group. Four (23.5%) and 3 (9%) cows of the IFI and IUI groups, respectively, became pregnant. On days 8-11 post-insemination, ovulation failure was registered in 3 (18%) and in 4 (12%) cows of the IFI and IUI groups, respectively. Our results suggest that IFI could be used as an alternative procedure to the usual deposition of semen into the uterus in cows of low fertility.

Sperm head binding to epithelium of the oviduct isthmus is not an essential preliminary to mammalian fertilization - review.

In endeavouring to understand the nature of sperm-oviduct interactions in mammals, attention was focused on experimental models in which fertilization can occur without a preliminary phase of sperm head binding to the isthmus epithelium. The ovarian endocrine milieu imposed on the oviduct tissues plays an important role in the binding phenomenon, although less so after the time of ovulation. Nonetheless, a sperm suspension introduced into the peritoneal cavity or surgical insemination directly into the oviduct ampulla before ovulation can result in fertilization, as can a surgical model in which the isthmus has been resected and the remaining portions of the duct reanastomosed. Mating or artificial insemination after ovulation in pigs permits rapid sperm transport to the site of fertilization, and the frequency of polyspermic penetration increases with the post-ovulatory age of eggs.Strategies underlying sperm binding were considered, especially in terms of preovulatory sperm storage and suppression of full membranous maturation. These, in turn, raised the problem of how sperm binding in vitro to oviduct cells from prepuberal animals or to cells harvested during the luteal phase of the estrous cycle, or to cells from the ampulla or even the tracheal epithelium, can act to regulate sperm storage and maturation with precision. In an evolutionary perspective, preovulatory binding of diverse populations of cells to the endosalpinx may have developed as a form of fine tuning to assist in sperm selection, to synchronize completion of capacitation with the events of ovulation, and to promote monospermic fertilization by a controlled release of competent gametes.

When is a cow in estrus? Clinical and practical aspects.

Good detection of estrus is critically important in dairy husbandry. Incorrect detection of estrus is related to loss of profit due to extended calving intervals, milk loss, veterinary costs, etc. Detection of estrus remains a major problem despites enormous progress in the knowledge of reproductive physiology of the cow and in development of estrus detection aids. To achieve good estrus detection, many factors have to be taken into account. On one hand a cow has to express estrus and on the other hand the farmer has to detect it. Combined action of several hormones causes physiological changes that lead to ovulation and an environment in the uterus that allows sperm to fertilize the egg. Besides these internal actions, a number of external changes can be observed. When using visual observations, time of the day and time spend on observation have a great impact on detection rates. Many devices are available to aid in estrus detection, such as pedometers, mount devices, temperature, and hormone measurements. Expression of estrus can be influenced by many factors. Heritability, number of days postpartum, lactation number, milk production, and health are known to influence estrus expression. Environmental factors like nutrition, season, housing, herd size, etc. also play a role in estrus expression. To evaluate estrus detection, record keeping is very important; a number of formulas can be used to assess detection efficiency. Besides the farmer, the veterinarian and inseminator can play an important role in estrus confirmation and good insemination strategy. In the end, the time of ovulation and the age of the egg at sperm penetration is critical for conception. Therefore, emphasis in research needs to be on the timing of insemination relative to ovulation, and thus on the detection of ovulation.

Does heat stress provoke the loss of a continuous layer of cortical granules beneath the plasma membrane during oocyte maturation?

The objective of the present study was to evaluate the influence of heat stress on bovine oocyte maturation. Both nuclear stage and distribution of cortical granules (CG) were simultaneously evaluated in each oocyte. Oocyte overmaturation under standard conditions of culture was also evaluated. For this purpose, logistic regression procedures were used to evaluate possible effects of factors such as heat stress, overmaturation, replicate, CG distribution and metaphase II (MII) morphology on oocyte maturation. Based on the odds ratio, oocytes on heat stressed (HSO) and overmaturated (OMO) oocyte group were, respectively, 14.5 and 5.4 times more likely to show anomalous MII morphology than those matured under control conditions (CO). The likelihood for an oocyte of showing the CG distribution pattern IV (aging oocyte) was 6.3 and 9.3 times higher for HSO and OMO groups, respectively, than for the CO group. The risk of undergoing anomalous oocyte maturation, considering both nuclear stage and distribution of CG was 17.1 and 18 times greater in oocytes cultured in HSO and OMO groups, respectively, than those in the CO group. In conclusion, heat stress proved to be valuable in aging oocytes. Heat stress advanced age for nuclear and cytoplasmic processes in a similar form to that of oocyte overmaturation.

Factors affecting spontaneous reduction of corpora lutea and twin embryos during the late embryonic/early fetal period in multiple-ovulating dairy cows.

Spontaneous reduction of advanced twin embryos has been described in high-producing, Holstein-Fresian (Bos taurus) dairy herds. The first objective of the current study was to determine whether management and cow factors could have an effect on such a reduction in twin pregnancies during the early fetal period. Because loss of a corpus luteum was noted in cows suffering twin reduction, we expanded our study to include multiple-ovulating cows carrying singletons. Pregnancy was diagnosed and confirmed from Days 28 to 34 and 56 to 62 postinsemination. Sixty-nine (23.5%) of 293 pregnant cows with two corpora lutea carrying singletons and 132 (28.4%) of 464 twin pregnancies recorded on first pregnancy diagnosis subsequently lost one of the corpora lutea or one of the embryos, respectively. Thirty-four (25.8%) of the 132 twin pregnancies suffering embryo reduction lost one corpus luteum along with the embryo. Corpus luteum reduction always occurred in the ovary ipsilateral to the gravid horn suffering embryo reduction. Binary logistic regressions were performed considering corpus luteum and embryo reduction as dependent variables in single and twin pregnancies, respectively, and several management- and cow-related factors as independent variables. In cows carrying singletons, the risk of corpus luteum reduction was 14.3 (1/0.07) times lower for a given herd, whereas the interaction season by laterality significantly affected corpus luteum reduction such that in cows with two corpora lutea ipsilateral to the horn of pregnancy, the risk of reduction decreased during the winter period. In cows carrying twins, ipsilateral twin pregnancies were 3.45 (1/0.29) times more likely to undergo the loss of one embryo than bilateral twin pregnancies. As an overall conclusion, both corpora lutea and embryos were vulnerable to the effects of stress factors during the early fetal period in cows maintaining their pregnancies. A strong unilateral relationship between the corpus luteum and the conceptus was also observed.

Differing sperm ability to penetrate the oocyte in vivo and in vitro as revealed using colloidal preparations.

The penetration ability of boar (Sus scrofa domestica) spermatozoa exposed to viscous preparations under in vivo and in vitro fertilization conditions has been examined. Experiments involving induced ovulation in prepubertal animals and surgical insemination directly into the oviduct isthmus revealed an advantage of colloidal preparations. Based on within-animal comparisons, the incidence of penetration was 100% using both spermatozoa suspended in a viscous preparation of plant extracts and spermatozoa suspended in a control medium. However, percentages of monospermy were 22.2% in 54 oocytes inseminated with the control suspension compared with 62.5% in 48 oocytes inseminated with the colloidal preparation. An in vitro study involving 355 oocytes from slaughterhouse ovaries inseminated with in vitro-capacitated spermatozoa gave similar percentages of penetrated oocytes for both the control and colloidal suspensions. In this case, however, the percentage of monospermy was 32.7% in the control group compared with 10.6% for spermatozoa suspended in the colloidal preparation. Higher mean numbers of sperm inside the oocytes and higher numbers of sperm bound to the zona pellucida were also observed with the colloidal suspensions. In vitro motility and viability for spermatozoa in the colloidal suspensions were enhanced compared with that of the control group. Lower sperm membrane lipid disorder and reactive oxygen species generation were also observed in the viscous solution. These findings suggest that viscous fluids can enhance the ability of sperm to move, bind, and penetrate the oocyte in vitro, although this influence may be masked in vivo due to the already high viscosity in the oviductal fluid close to the time of ovulation.

Anomalous pregnancies during late embryonic/early foetal period in high producing dairy cows.

This study analyses anomalous cases of gestation ending in pregnancy loss during the early foetal period and their effect on progesterone and plasma pregnancy-associated glycoprotein-1 (PAG-1) concentrations. Data derived from a large-scale ultrasound pregnancy diagnosis programme in high producing dairy cows. Over a 3-year period (2004-2007), a very low incidence (0.5%: 15 of 3094) of anomalous pregnancies was recorded. The results revealed that the following anomalies were detected on days 35-41 of gestation in cows carrying singletons with one single corpus luteum: embryo death in eight cows (0.3%); and embryo in the uterine horn contralateral to the corpus luteum in seven cows (0.2%). All these animals suffered pregnancy loss during the early foetal period. In cows carrying dead embryos, no signs of conceptus degeneration were observed on pregnancy diagnosis. Amnion size (approximately 25 mm diameter) and uterine horn fluid contents were estimated to be similar to those of the normal pregnant cows in this period. In the contralateral gestations, live embryos were observed in all ultrasound checks before pregnancy loss. Uterine fluid contents increased in the two cows in which gestation continued for more than a week. In the cases of embryo death but not in those of contralateral gestation, a drop in PAG-1 levels was noted prior to pregnancy loss. Two cows carrying dead embryos increased with time allantoic fluid contents. The PAG-1 values increased with time in one cow bearing a dead embryo (from 2.31 to 6.79 ng/ml) and in two of the contralateral gestations (from 1.66 to 2.33 ng/ml and from 0.39 to 6.79 ng/ml, respectively). Results of this study indicate that the foetal membranes continue to undergo some activity following embryo death, and that contralateral pregnancy may determine failure of the gestation process.

Sperm release from oviduct epithelial binding is controlled hormonally by peri-ovulatory graafian follicles.

To avoid inappropriate conclusions being drawn from the extensive use of in vitro preparations of sperm-oviduct epithelial binding, it is recalled that events in the genital tract of mammals are regulated by the gonads, primarily by their changing secretion of steroid hormones. Key observations from in vivo models are used to emphasise the dynamic interactions between viable sperm cells and the caudal (distal) portion of the oviduct isthmus, the site of the functional sperm reservoir. These include (1) pre-ovulatory arrest and epithelial binding of intact sperm cells and thereby suppression of completion of capacitation, (2) peri-ovulatory activation and release from binding of discrete sub-populations of competent spermatozoa, and (3) post-ovulatory liberation of large numbers of spermatozoa. These observations underline the influence of endocrine regulation of sperm binding and release by peri-ovulatory Graafian follicles, a point brought out by the enhanced sperm release prompted by diverse treatments with solutions of progesterone. In the light of this evidence, the suitability of in vitro preparations for clarifying physiological events should be questioned, especially if myosalpingeal catecholamines diffusing out of the autonomic nervous system contribute to sperm activation and/or release. None of this is to infer that sperm cells themselves are without influence on their epithelial binding reaction(s). Nor is it to suggest that in vitro models of sperm-oviduct binding are without relevance to the development of sperm evaluation technologies. However, pre-ovulatory sperm-epithelial binding and a regulated peri-ovulatory release should be seen as vital tactics in the overall strategy of achieving successful monospermic fertilisation.

The peritoneal mesothelium covering the genital tract and its ligaments in the female pig shows signs of active function.

The aim of this study was to describe the surface features of the peritoneal mesothelium covering the genital tract and adjacent ligaments of the sow to find signs of biosynthetic activation of cells. Surface features of the serosa covering the genital tract and adjacent ligaments from 14 cyclic sows, 7 in the follicular phase and 7 in the luteal phase of the estrous cycle, were examined by histology and scanning electron microscopy. Five additional sows, three in the follicular phase and two in the luteal phase of the estrous cycle, were examined by transmission electron microscopy (TEM). In this study, the presence of cells of the oviductal epithelium in the serosa of the infundibulum and the ampulla, as well as indications of a high functional activity of the mesothelial cells in the areas studied were two aspects that differed from the findings of previous works. Presence of endosalpingeal cells was observed in the serosal surface, showing cyclical variations with a predominance of either ciliated cells during the follicular phase or secretory cells during the luteal phase. Signs of high functional activity of the mesothelial cells included the predominance of cuboidal over flattened cells, a cytoplasm richly supplied with organelles, a dense microvillous coat, numerous primary cilia, and many secretory structures on the surface of cells. These results indicate that the serosa covering the genital area and the adjacent ligaments in the sow has an active epithelium whose coordinating role between reproductive tissues may be far more significant than previously thought.

Peritoneal fluid as an unrecognised vector between female reproductive tissues.

This essay discusses the potential contribution of peritoneal fluid to the regulation of reproductive processes in female mammals. After noting the relatively high concentrations of diverse hormones in peritoneal fluid, and accepting that it bathes the surface of all the internal reproductive organs, peritoneal fluid is then proposed as a means of communication between the two ovaries. It could act to influence both the hierarchy of Graafian follicles and the rate and extent of development of a newly-formed corpus luteum. Cytokines in peritoneal fluid are considered in this context, as are the differing populations of leukocytes. Circumstantial evidence is offered for entry of peritoneal fluid into the Fallopian tubes, especially close to the time of ovulation, with the suggestion that such entry could modify the nature of endosalpingeal secretion and transudation. Thereafter, the spectrum of gonadal hormones in peritoneal fluid could be influencing uterine tissues in a local manner. Finally, reference is made to regional differences in the concentration of steroid hormones in the peritoneal fluid of women, likewise to regional differences in domestic animals.