Whitten Effect Research Articles

Influencing and Regulating the Estrous Cycle in Outbred CD1 Laboratory Mice (Mus musculus)

AbstractThe Whitten effect is a widely used tool for manipulating the mouse estrous cycle and generating reproductively active females within the laboratory setting. Typically, peak numbers of sexually receptive mice occur following exposure to male pheromones, resulting in a higher number of successful copulations on the third day after exposure. Although this method has improved efficiencies, the percentage of females mated and subsequently deemed to be pregnant/pseudopregnant remains relatively low, around 50%. In experiment 1, we aimed to 1) further understand cyclicity; 2) determine whether the initial cycle stage plays an importance on day 3 receptivity; and 3) identify any repetitive patterns/cycle stabilization. Mice (n = 27) were assigned to group cages according to cycle stage (proestrus, estrus, metestrus, diestrus). Experiment 2 was developed to determine an optimum treatment to promote receptivity by exposure to various pheromone stimuli. Mice (n = 45) were randomly assigned to 5 treatment groups (PBS-treated sham soiled bedding, male soiled bedding, live male, pregnant females, and lactating females). In both experiments, daily vaginal cytology was performed for 21 days to determine the cycle stage. Results from experiment 1 indicate that the initial cycle stage did not contribute to day 3 receptivity, although synchronization within several groups/cages was noted, and that the greatest numbers of estrous animals were obtained on days 6 and 7. Experiment 2 revealed that exposure to live males and lactating females both significantly improved receptivity compared with the PBS, male soiled bedding, and pregnant female groups. These results indicate that current strategies used for routine synchronization could be further improved through alternative housing regimens without compromising animal welfare.

THE EFFECT OF 4-VINYLCYCLOHEXENE DIEPOXIDE ON FEMALE NUTRIA (MYOCASTOR COYPUS) FERTILITY IN CAPTIVITY-A PILOT STUDY.

The nutria (Myocastor coypus) is a globally widespread invasive species. Attempts to eradicate nutria by shooting, poisoning, and trapping have been mostly unsuccessful, leading to calls for the development of new control methods. The compound 4-vinylcyclohexene diepoxide (VCD) is known to cause follicular atresia in mammals and may control conception when administered orally. It was hypothesized that VCD administered PO will cause follicular destruction in female nutria. VCD (250 mg/kg PO) was administered or coconut oil, as a control, to five nutria females each for 12 d. Sixty days following VCD exposure, males were introduced to the females. Over the following 7 mon, the effect of VCD on nutria fertility was assessed by conducting ultrasound monitoring to determine pregnancy status and measuring blood serum progesterone and estradiol levels. Finally, after performing ovariectomies, viable follicles were counted on histologic ovarian cortical sections. It was found that the female estrous cycles became synchronized, suggesting a Whitten effect in this species. Also, an increase in the females' serum progesterone levels following the introduction of males occurred, suggesting a male presence effect. Orally administered doses of 250 mg/kg VCD for 12 d had no significant effect on nutria pregnancy rates or on the number of follicles in the ovaries examined. Further studies, using a higher dose or longer administration period, are necessary to conclude whether orally administered VCD can be used as a contraceptive agent for nutria.

Male scent but not courtship vocalizations induce estrus in wild female house mice

Exposure to males or male urinary scent can induce and accelerate the rate of female estrous cycling in house mice ("Whitten effect"), and this response has been replicated many times since its discovery over 60 years ago. Here, we tested whether exposing female mice to recordings of male courtship ultrasonic vocalizations (USVs) induces estrous cycling, and whether exposure to both male scent and USVs has a stronger effect than to either of these stimuli alone. We conducted our study with 60 wild-derived female house mice (Mus musculus musculus). After singly housing females for 14 days, we monitored estrous stages via vaginal cytology for two weeks while isolated from males or male stimuli. We continued monitoring estrus for two more weeks during experimental exposure to one of four different types of stimuli: (1) clean bedding and background noise playback (negative control); (2) recordings of male USVs (16 min per day) and clean bedding (male USV treatment); (3) soiled male bedding and background noise playback (male odor treatment; positive control); or (4) male USVs and soiled male bedding (male odor and USV treatment). Females were then paired with males to test whether any of the four treatments influenced female reproduction (especially latency to birth). We confirmed that exposure to male odor increased female cycling, as expected, but exposure to recordings of male USVs had no effect on estrus. Females exposed to both USVs and odor went through more cycles compared to controls, but did not differ significantly from exposure to male odor (and background noise). After pairing females with a male, females showing male odor-induced cycling produced their first litter sooner than controls, whereas USVs did not have such an effect. This is the first study to our knowledge to show that male odor induces estrus in wild house mice and to show functional effects on reproduction. Our results do not support the hypothesis that male vocalizations induce female estrus, although we suggest other approaches that could be used to further test this hypothesis.

Mating success of timed pregnancies in Sprague Dawley rats: Considerations for execution.

This study compares three different mating techniques in Sprague-Dawley rats, using the pregnancy rate as the main indicator of success. It provides recommendations for timed-pregnancy experiments to achieve an appropriate sample size for the study of human pregnancy disorders. The implementation of a preconditioning phase, determination of the estrous cycle, the use of two mating strategies (Lee-Boot and Whitten effect), female: male mating ratios, and cohabitation duration should be considered as they improve the mating success rate.

Effect of Xylene Oral Exposure on the Mouse Uterus – A Preliminary Study

Abstract Xylene is one of the environmental pollutants with a negative impact mainly on several organ systems. The aim of this study was to determine the effect of xylene on the uterus of mice. The study was performed on 12 adult female mice. Control mice (n = 6) were fed shredded pellets at a dose of 4 g per day. Xylene mice (n = 6) were fed the same diet at the same dose and orally administered xylene at 10 μl per day for 14 days. The mice were synchronized using the Whit-ten effect and introduced to males before the end of the procedure. Mice of both groups with no copulation plug were euthanized by cervical dislocation. The uteri were collected for routine histological and immunohistochemical analysis. The endometrial epithelium demonstrated vacuolar degeneration, mitotic cell activity, and the presence of leukocytes typical of metoestrus. Reductions of the endometrium, stroma, and myometrium were observed in the xylene mice. The xylene application did not have a significant effect on the superficial epithelium, or the size and number of uterine tubular glands. The immunohistochemical analysis of a proliferation marker PCNA revealed that the xylene increased its expression in the stroma, endometrial and myometrial cells, but did not significantly affect the superficial epithelial cells. The expression of an anti-apoptotic marker Bcl-xl in the xylene mice was stronger in the superficial epithelial, stromal, and endometrial cells. The Bcl-xl expression in the myometrial cells was similar to the controls. The results showed that the application of xylene stimulated the proliferation and exerted an anti-apoptotic effect on the uterine cells. However, the increased proliferation can lead to the malignant transformation of cells, resulting in their uncontrollable division.

Pseudopregnancy and reproductive cycle synchronisation cannot be induced using conventional methods in the spiny mouse (Acomys cahirinus).

The menstruating spiny mouse is the first rodent identified to exhibit natural spontaneous decidualisation, cyclical endometrial shedding and regeneration. While the spiny mouse shares several primate-like characteristics in its reproductive biology, it has not been established whether pseudopregnancy can be induced or if its cycles can be synchronised as in non-human mammals. Here we describe attempts to induce pseudopregnancy and synchronisation of menstrual cycles (i.e. Whitten effect) in spiny mice. Virgin females (n=3-8 per group) underwent one of the following procedures to induce pseudopregnancy: daily vaginal lavage only (control), progesterone injection, mechanical stimulation of the cervix and sterile mating. A separate cohort was also exposed to male-soiled bedding to assess the Whitten effect. Pseudopregnancy was deemed successful if females presented with extended (>12 consecutive days) leukocytic vaginal cytology. No female from any method of induction met this criterion. In addition, the menstrual cycles of a group of six females could not be synchronised, nor immediate ovulation induced via exposure to male-soiled bedding. These responses indicate that the spiny mouse does not behave as a typical rodent. Like higher-order primates, the spiny mouse exhibits a relatively rare reproductive strategy, of failure to show pseudopregnancy or cyclical synchronisation. This is further endorsement of the use of this species as a versatile animal model for translational studies of menstruation and fertility.

The Use of Natural Phenomenon in Obtaining Pregnant Rats and Mice as Experimental Animals with The Same Gestational Ages

Hormonal oestrus synchronization is often used to obtain the same gestational ages animal models, but it may not physiologic nor practical. This research was conducted to find out a simple method in obtaining physiologic pregnant experimental animals. We used 120 female rats and mice each, divided into 16 groups with 15 members each. They were classified according to the state of parities, pair or group mating, and synchronization of oestrous. The estrous synchronization is done by utilizing the natural phenomenon (Lee-Boot, Pheromone, and Whitten effect). All nulliparous and multiparous models then were simultaneously mated for one night in pairs (1:1) or in groups (1:5) subsequently. Pregnancy rates were noted based on the number of models delivering pups on day 19-23 post-mating. The highest pregnancy rate was noted in group of multiparous, synchronized and monogamous rats (86.7%), followed by nulliparous, synchronized and monogamous rats (73.3%) and multiparous, synchronized and monogamous mice (60%) (p<0.05). Synchronization of the estrous and monogamous mating of rats or mice had significant roles in pregnancy rates (p<0.05). To get a high number of pregnant rats or mice models, we suggest to use the multiparous, estrous synchronized and monogamous simultaneous mating within one night.

Determination of the whitten effect based on vaginal cell characteristics, vulva appearance and behavior in grouped female mice

Introduction: Pheromones are chemicals produced by an animal that affects the behavior of another animal or the same species. Information conveyed includes location, presence of food or threat, sexual attraction, courtship, and dam–pup interactions. Pheromones are used widely in laboratory mice facilities to synchronize estrus and simultaneous breeding for logistic purposes. Female mice housed together in the absence of the male exhibit the Lee-Boot effect of lengthened diestrus or ovarian inactive period of up to several weeks. Whitten effect is described when a large number of female mice housed together in the absence of the male and having diestrus, will enter estrous 48 to 72 hours later upon exposure to male odors or male mouse urine soaked-bedding. Objective: The aim of this study is to determine the time taken for the Whitten effect to occur based on changes in vaginal cell characteristics, vulva appearance and behavior in grouped female mice. Methodology: Ten female mice were acclimatized to the animal facility for 3 estrus cycles or 12 days. Phases of the estrus cycle were evaluated by visual observation to assess changes to the vulva and vaginal cytology. Male urine soaked-bedding were exposed to females for 3 days and the time taken for the Whitten effect to occur was determined based on changes in vaginal cell characteristics, vulva appearance and observation of behavior. Result: The Cochran's Q test was used to observe the changes from diestrus to proestrus and later estrus. The results showed a significant difference (p<0.05) in the number of mice that successfully enter the proestrus and estrus phases over a four time point, ?2 (2) = 18.857. Conclusion: The Whitten effect occurs after 72 hours of exposure to male urine soaked-bedding based on vulva appearance, vaginal cell characteristics and behavior in grouped female mice.

Occlusion of Seminal Vesicles Increases Sexual Activity in a Mouse Model

BackgroundLittle is known about the physiologic role of seminal vesicles beyond their fertility function. It has been suggested repeatedly that seminal vesicles have an impact on sexual activity. Although this has been investigated in various animal models, such a role has never been found. ObjectiveTo assess in a novel mouse model whether occlusion of seminal vesicles affects sexual activity. Design, setting, and participantsAdult male CD1 mice (n=77) were assigned randomly to the experimental groups: (1) seminal vesicle occlusion (SVO) (n=24), (2) seminal vesicle resection (SVR) (n=23), and (3) sham operation (SO) (n=30). Adult females were brought into estrus by the Whitten effect. After recuperation, mouse pairs were observed during sessions of 3h each. Sexual activity was analyzed separately by three observers blinded to the experimental conditions. InterventionSVO, SVR, and SO. Outcome measurements and statistical analysisThe primary end point was percentage of sessions with intromission; secondary end points were number of intromissions and latency until first intromission. A logistic regression model and the Kruskal-Wallis test were used. Results and limitationsA total of 141 sessions for a total of 423h were analyzed. Intromission was scored in 20 of 42 sessions (48%) with SVO mice, a significantly higher rate than the 8 of 39 sessions (21%) with SVR mice (p=0.001) and 18 of 60 sessions (30%) with SO mice (p=0.004). Secondary end points were comparable in all three groups (p=0.303 and 0.450, respectively). ConclusionsMales with SVO were significantly more often sexually active than males undergoing SVR or SO. This suggests that occluded, and thus engorged, seminal vesicles increase sex drive in male mice. Since the potential clinical benefit might be highly relevant, further studies should confirm these promising results and investigate the potential application in men.

Recent Advances in Reproductive Technologies

Never before have there been so many medical intervention options available to infertile couples seeking to become parents. These options are possible largely due to the research in animal reproduction which started several decades ago with the widespread use of artificial insemination in farm animals. This was followed by the advent of in vitro embryo production/manipulation and transfer, cryopreservation, and the development of reproductive cloning (that sparked an unprecedented interest in stem cell technology). In addition to advancing our ability to modify reproductive function and changing our perception of what is possible, such technologies have also greatly expanded our understanding of reproductive/gamete biology. We have solicited research and review articles related to the recently developed technologies in animal reproduction for this special issue of the journal. The response was overwhelming and a number of exciting articles have been selected for publication. Most of the accepted papers in this issue are review papers (9 of 15) and the articles have been contributed by researchers from a dozen different countries, reflecting its international scope. We are extremely grateful to all the reviewers who took time to carefully read the submitted manuscripts and to provide critical comments which helped to ensure the high quality of this issue. First few papers in this issue deal with sperm/semen. Morrell & Rodriguez-Martinez summarize the recent advances in sperm selection and its application in reproductive biotechnologies, including fractionated semen collection, cryopreservation, biomimetic sperm selection, sperm sex selection, and hyaluronic acid binding selection. Bansal & Bilaspuri review the impacts of oxidative stress and reactive oxygen species on sperm/semen function and highlight the emerging concept of utilizing oxidative stress as a tool for contraception. Rodriguez-Martinez & Wallgren review the new developments in cryopreservation of boar semen, a species from which the semen is known to be difficult to cryopreserve. Caballero et al. overview the importance of sperm interaction with the male reproductive fluids to acquire the fertilization ability and highlight the role of membranous vesicles (epididymosomes and prostasomes) present in these fluids. The research article by Mollineau et al. presents the results of comparing a number of semen extenders and storage options on sperm motility of agouti, a neotropical edible rodent. Next few papers in this special issue address the transplantation of gonadal tissue and cells. Honaramooz & Yang provide a review of the salient recent research on germ cell transplantation in farm animals with emphasis on examination of ways to increase its efficiency through improved preparation of the recipient testes as well as isolation, purification, preservation, and transgenesis of the donor germ cells. The review paper by Mota et al. highlights those aspects of testis tissue xenografting that need further attention, namely, determinants of its outcome, preservation of the donor tissue, and subsequent ART techniques to produce offspring from the recovered material from grafts. The research paper by Abbasi & Honaramooz presents the results of a study aimed at improving the outcome of testis tissue xenografting by comparing the effects of using different numbers of donor tissue fragments to be grafted. In the next few papers in this issue, oocytes/follicles have been discussed. Prentice & Anzar provide a review of the recent advances in cryopreservation of mammalian oocytes as a way to bank ova using slow freezing and vitrification. The research article by Presicce et al. presents the results of a study aimed at investigating the efficiency of in vitro embryo production based on the source of oocytes (recovered by ovum pick-up), hormonal stimulation, and utilizing sexed bovine sperm. Andrade et al. present their research results in which they tested the effects of culture media and supplements as well as fragments of cultured tissue on the ultrastructure of ovine primordial follicles. The last few papers in this issue of the journal relate to a variety of subjects and techniques in reproduction. Monteiro et al. review the factors involved in nuclear reprogramming and discuss in vitro manipulations that potentially reduce epigenetic errors and better mimic in vivo conditions for preimplantation bovine embryos. The review article by de Sa Fihho et al. summarizes the current hormonal treatment protocols, as well as other approaches, used to optimize the reproductive performance of beef cattle reared under tropical environments. Buranaamnuay et al. provide their research findings on the comparison of two methods of artificial insemination (intrauterine versus deep intrauterine) in sows using frozen-thawed boar semen on fertilization rate and number of embryos. The last paper in this issue is a research article by Booth & Webb reporting on the effects of blockage of the vomeronasal organ ducts on serum concentrations of LH in South African does while exposed to bucks (Whitten effect). A. Honaramooz S. Schlatt K. Orwig N.-H. Kim

Effect of Blockage of the Ducts of the Vomeronasal Organ on LH Plasma Levels during the “Whitten Effect” in Does

Eighteen mature, nonpregnant, and indigenous South African does were randomly divided into two groups to test if their vomeronasal organs exert an influence on LH plasma levels during a Whitten effect experimental trial. Does in the treatment (VNO ablated) group had their vomeronasal organs rendered nonfunctional by cauterization of the nasoincisive duct under surgical anesthesia. Does in the control group had their nasal civities irrigated with physiological saline under surgical anesthesia. All does were synchronized into oestrus and introduced to bucks one day prior to their expected second oestrus cycle. Successful matings were recorded. Timely blood samples were collected during each of the five days before and five days after buck introduction. Blood plasma concentrations of estradiol and LH were determined by radioimmunoassay. Analysis of variance between groups demonstrated that the does in the VNO ablated group did not demonstrate any interest in mating, did not become pregnant, and did not demonstrate the primary increase in tonic plasma levels of LH that is necessary for ovulation to occur. By contrast, all of the does in the control group demonstrated successful matings, became pregnant, and demonstrated typical primary tonic level increases and preovulation surges in LH. Thus, it was concluded that the vomeronasal organ modulates the primary increase in tonic levels of LH and thus influences ovulation that occurs during the Whitten effect in South African indigenous does.

Positive identification of the puberty-accelerating pheromone of the house mouse: the volatile ligands associating with the major urinary protein.

Five structurally diverse small ligands, all binding to the major urinary protein (MUP) of the male house mouse, show individually puberty-accelerating pheromonal activity in the recipient females. A recombinant MUP (identical structurally to the natural protein) has shown no biological activity. While four of these ligands were previously implicated in oestrus synchronization (Whitten effect), the same chemosignals now appear responsible for both sexual maturation and cycling in adult females.

Sensitivity to diethylstilbestrol-(DES)-induced abortions is influenced by the whitten effect

Group-housed female mice, when introduced to a male, will undergo a synchronized estrous cycle with a characteristic pattern of mating where the majority of animals mate on the third day. This phenomenon is termed the Whitten effect. Exposure of pregnant mice to diethylstilbestrol (DES), a potent nonsteroidal estrogen, is capable of inducing premature parturition. The purpose of this study was to evaluate abortion induced by a single injection of DES on day 15 of gestation and compare the sensitivity depending on the day of mating. Initial observations confirmed that those animals that mated on day 3 were significantly more susceptible to DES-induced abortion than animals that mated on any other day. The possibility that this increase in sensitivity to abortion may be the result of differences in number or size of embryos at the time of DES exposure was examined. No significant difference was observed in the number of embryos at day 4 of gestation, at day 15 of gestation, or at birth. There was no difference in the weight of embryos at day 15 of gestation or at birth. A significant increase in the ratio of embryos between uterine horns was observed in the animals that mated on day 3 (ratio = 2.13) compared to animals mating on other days (ratio = 1.23 to 1.48). The cause of increased sensitivity to DES in animals that mated on day 3 is unclear. Our results may indicate that the induction of ovulation associated with the Whitten effect causes unequal embryo distribution leading to functional differences in sensitivity of the pregnancy to exogenous stimuli. It is evident that it may be critical to control for the time of mating in toxicologic studies.

Investigations on the Olfactory Discrimination Exhibited by the Females in the Male‐induced Implantation Failure (the Bruce Effect) in Laboratory Mice

AbstractDirect contact with the alien male pheromone for 7 days before mating significantly decreased the rate of male‐induced implantation failure (the Bruce effect) in newly inseminated female mice. By contrast, females housed with alien males for 7 days before mating so that physical contact with the latter was prevented exhibited a high rate of implantation failure following exposure to alien males after mating. Exposure to an air‐borne pheromone produced by alien males induced oestrus (the Whitten effect) in a significant number of grouped females. The results do not support the suggestion that the inability of the stud male to block implantation is due to his original induction of oestrus. The results also do not support the view that the key factor in the induction of the Bruce effect is the ability of the female to identify the stud as an individual which prevents her from responding to him but allows her to react to a new male with hormonal changes leading to implantation failure and return to oestrus. It is suggested that the olfactory discrimination exhibited by the female in the Bruce effect is influenced by her overall olfactory experience before and after mating. The results are consistent with the view that the male‐originating pheromone involved in the Bruce effect is distinct from the one involved in the Whitten effect.

Promotion of the Whitten effect in female mice by synthetic analogs of male urinary constituents.

Two volatile constituents of male mouse urine, 2-(sec-butyl)-4,5-dihydrothiazole and dehydro-exo-brevicomin, were synthesized and tested for their ability to induce estrous cycle in female mice (the Whitten effect). The suppression of ovarian cycling activity that resulted from grouping the females was abolished by exposure to normal male urine. The synthetic compounds, when added together in appropriate concentrations to the (previously inactive) urine of castrated males, or even to water, were found to be as effective as normal male urine. The action of the synthetic compounds appears attenuated for singly caged females.

Evaluation of the involvement of the vomeronasal organ in the pheromonal influences on the estrous cycle of the laboratory mouse

The estrous-cycle irregularities induced in female mice by unisexual grouping were not abolished by ablation of the vomeronasal organ (VNO). The cycle irregularities in VNO-ablated females housed in unisexual groups were similar to those in sham-operated and intact females housed in unisexual groups. Exposure of unisexually grouped VNO-ablated females to conspecific males induced estrus in the majority of females within 7 days with the characteristic peak on day 3 (the Whitten effect). Sham-operated, as well as intact females also showed the Whitten effect following male exposure. The results suggest that the VNO is not involved in the perception of the female-originating pheromone inducing estrous-cycle irregularities, and the male-originating pheromone causing induction of estrus.

Studies of the male-originating pheromones involved in the Whitten effect and Bruce effect in mice.

Experiments were designed to elucidate the mode of transmission of the male-originating pheromones involved in the induction of estrus (the Whitten effect) and in implantation failure (the Bruce effect) in mice. The Whitten effect was induced in unisexually grouped females by exposure to corralled males, and also by corralled males housed within a perforated cage (which prevented physical contact of the females with the male-originating pheromone). The results suggest that the pheromone involved in the Whitten effect is volatile (airborne). Implantation failure occurred in a significantly high proportion of newly inseminated females when they were individually confined in corrals and housed below corralled alien males. By contrast, implantation failure was significantly reduced when corralled females were housed above corralled alien males. The results indicate that the male-originating pheromone involved in the Bruce effect is nonvolatile and acts on the females through contact. It is suggested that the pheromone involved in the Whitten effect is distinct from the one involved in the Bruce effect.

Oestrus induction in unisexually grouped mice by multiple short-term exposures to males.

Oestrus induction and synchronization (the Whitten effect) were achieved in unisexually grouped female mice by short-term (10 and 30 min) exposure to conspecific males.

Absence of sexual imprinting in house mice cross-fostered to deermice

Female albino house mice ( Mus musculus, SJL/J strain) were cross-fostered within 0 to 12 hours of birth to lactating female prairie deermice (Peromyscus maniculatus bairdii) and their mates. After weaning at 18 days, the cross-fostered females were subjected to a series of physiological and behavioral tests designed to examine the species specificity of response. The physiological tests employed were: (1) male-induced puberty (the Vandenbergh effect), (2) mating synchrony (the Whitten effect), and (3) pregnancy block (the Bruce effect). Behavioral responses were tested in a T maze by measuring the amount of time spent investigating soiled bedding or confined males of each of the two species during a 30 min period. Both physiological and behavioral tests indicate that raising female house mice with deermice: (1) does not cause them to imprint on deermice and (2) does not prevent their responding to house mouse males. Therefore, species specificity of pheromonal responses and preference behavior in female house mice appears to be genetically programmed instead of imprinted.

Nidation induced in mice during the lactational delay by the presence of strange males.

In a previous study (Bloch, 1971) we have suggested that the block to pregnancy brought about in newly mated mice by the odour of strange males, the 'Bruce effect' (Bruce, 1960), is provoked by a secretion of follicle-stimulating hormone (FSH) and, hence, oestrogen. This secretion inhibits nidation in non-lactating females, but hastens implantation in suckling animals, thus enhancing the occurrence of on-time nidations as was the case in our previous investigation. Oestrogen secretion would also agree with the 'Whitten effect'. Whitten (1956) established that females kept in groups without males and showing long periods of anoestrus showed synchronized oestrous cycles after the introduction of a male. The editor of Research in Reproduction (1971, Vol. 3, No. 4) commenting on our findings, writes: 'Dominic had earlier ascribed the pregnancy block to lack of prolactin and this important controversy on the effect of odours on the pituitary will be resolved only by