Effect of a partial deletion of Y chromosome on in vitro fertilizing ability of mouse spermatozoa.

The effect of a partial deletion of Y chromosome on sperm fertilizing ability was investigated through an in vitro fertilization technique. Epididymal spermatozoa of a congenic line, B10.BR-Ydel, which is characterized by a high incidence of abnormal spermatozoa, revealed a significantly lower in vitro fertilization rate (22%) than that (79%) of its control strain (B10.BR/SgSn), which has a normal-sized Y chromosome. Incidence of capacitated spermatozoa as determined by chlortetracycline fluorescence assay was significantly lower in B10.BR-Ydel than in B10.BR/SgSn spermatozoa. The fertilization rate was significantly improved when B10.BR-Ydel spermatozoa were separated from the supernatant of sperm suspension by Percoll gradient centrifugation. A reconstitution experiment revealed that the B10.BR-Ydel spermatozoa were more sensitive to the inhibitory effect of the supernatant than B10.BR/SgSn spermatozoa. Spermatozoa from F1 (C57BL/6N male x B10.BR-Ydel female) males showed higher fertilization rates than those from F1 (B10.BR.Ydel male x C57BL/6N female) males. These observations suggest that not only the morphology but also the fertilizing ability of spermatozoa is directly related to partial deletion of Y chromosome.     

Intrabursal transfer of spermatozoa (ITS): a new route for artificial insemination of mice.

Artificial insemination (AI) by direct injection of epididymal spermatozoa into the reproductive tract of females is simpler and more convenient than in vitro fertilization (IVF) and subsequent transfer of fertilized eggs to recipient oviducts for simultaneous acquisition of a large number of pups. Introduction of epididymal spermatozoa into oviducts via the oviductal wall or via vaginal and intrauterine routes is currently the most commonly used method for AI in mice. In this study, we explored another route for AI of the mouse and found that transfer of spermatozoa into a space near the infundibulum between the ovary and ovarian bursa enables in vivo fertilization of ovulated oocytes at the ampulla. When 1 microL of a sperm suspension containing 1 x 10(4) spermatozoa freshly isolated from B6C3F1 males was intrabursally injected into superovulated B6C3F1 females on E (embryonic day) 0.4 (10:00 AM), 5 of 7 females yielded 2-cell embryos with rates of efficiency ranging from 4 to 21% (11% on average), which were much lower than those (91% on average) for embryos obtained by natural mating. All the 2-cell embryos derived from injection of sperm developed in vitro to hatched blastocysts. Similar results were obtained from injection of 1 microL of sperm suspension containing 1 x 10(3) spermatozoa, although in vivo fertilizing ability was slightly improved (28% on average). When 1 microL of sperm suspension containing 1 x 10(4) spermatozoa was injected intrabursally into superovulated females that had been mated with vasectomized males, 6 of 10 mice (60%) yielded 19 normal mid-gestational fetuses with an average litter size of 3.2, which was much lower than that (14.5) for embryos obtained by natural mating. Although the present findings appear to be preliminary, this technique, based on the intrabursal transfer of spermatozoa, will be of practical use for AI in mice, particularly for transgenic and mutant mice that are often difficult to breed.     

An increased level of sperm abnormalities in mice with a partial deletion of the Y chromosome

Two congenic lines of mice, one with a partial deletion of the Y chromosome, differ in the percentage of spermatozoa with abnormal heads: B10.BR/SgSn males give 22.6% and B10.BR-Ydel/Ms males give 64.2% abnormal sperm. The F1s resulting from crosses of B10.BR/SgSn males with females of five common inbred strains exhibited significantly lower levels of abnormal sperm than the parental strains, as opposed to F1 hybrids sired by B10.BR-Ydel/Ms mutant males, where very high levels of abnormal spermatozoa were found. About 30% of abnormal spermatozoa, produced by males with deletion on the Y chromosome, were characterized by a flat acrosomal cap. This class of abnormality was never observed in non-mutant males, suggesting a mutant-specific defect. These results demonstrate the important role of the Y chromosome in spermatogenesis.     

Male sperm donation consequences in single and double matings in Anisopteromalus calandrae

Abstract. The number of spermatozoa that a male transfers to the female during copulation is a main component of its individual fitness, especially under the pressure of sperm competition. This paper presents experimental results on the direct relationship between the male's sperm investment and its paternity in the offspring of dual-mated females. An eye colour mutant (red-eyed) is used to study the differences in the mating and fertilization abilities of males through observation of single and dual matings of females in Anisopteromalus calandrae (Hymenoptera, Chalcidoidea, Pteromalidae). Experimentally, females accept dual matings only in the simultaneous presence of two males. Counts of spermatozoa in the seminal vesicles of virgin males show that red-eyed males have more sperm than wild-eyed ones (approximately 1.46-fold greater). Red- and wild-eyed males do not differ in their mating behaviour and females mate indifferently with both phenotypes. Compared with once-mated females, double-mated females increase neither sperm storage nor lifetime fecundity, and the offspring sex ratio is female-biased. Females mated with two males of different phenotypes produce offspring of both phenotypes throughout their reproductive life, whatever the order of males in the copulation sequence. Any mating pattern appears to produce more red- than wild-eyed offspring (between 1.45- and 1.88-fold greater). Thus, proportions of offspring of each male match the proportions of their sperm potential. With no preference of female for red-eye or wild-eye males being demonstrated at either behavioural or physiological levels, a male's investment in sperm quantity appears to determine its individual reproductive success, at least in these experimental conditions.     

Seminal proteins but not sperm induce morphological changes in the Drosophila melanogaster female reproductive tract during sperm storage

In most insects, sperm transferred by the male to the female during mating are stored within the female reproductive tract for subsequent use in fertilization. In Drosophila melanogaster, male accessory gland proteins (Acps) within the seminal fluid are required for efficient accumulation of sperm in the female's sperm storage organs. To determine the events within the female reproductive tract that occur during sperm storage, and the role that Acps and sperm play in these events, we identified morphological changes that take place during sperm storage in females mated to wild-type, Acp-deficient or sperm-deficient males. A reproducible set of morphological changes occurs in a wild-type mating. These were categorized into 10 stereotypic stages. Sperm are not needed for progression through these stages in females, but receipt of Acps is essential for progression beyond the first few stages of morphological change. Furthermore, females that received small quantities of Acps reached slightly later stages than females that received no Acps. Our results suggest that timely morphological changes in the female reproductive tract, possibly muscular in nature, may be needed for successful sperm storage, and that Acps from the male are needed in order for these changes to occur.     

Male Seminal Fluid Substances Affect Sperm Competition Success and Female Reproductive Behavior in a Seed Beetle

Male seminal fluid proteins are known to affect female reproductive behavior and physiology by reducing mating receptivity and by increasing egg production rates. Such substances are also though to increase the competitive fertilization success of males, but the empirical foundation for this tenet is restricted. Here, we examined the effects of injections of size-fractioned protein extracts from male reproductive organs on both male competitive fertilization success (i.e., P2 in double mating experiments) and female reproduction in the seed beetle Callosobruchus maculatus. We found that extracts of male seminal vesicles and ejaculatory ducts increased competitive fertilization success when males mated with females 1 day after the females’ initial mating, while extracts from accessory glands and testes increased competitive fertilization success when males mated with females 2 days after the females’ initial mating. Moreover, different size fractions of seminal fluid proteins had distinct and partly antagonistic effects on male competitive fertilization success. Collectively, our experiments show that several different seminal fluid proteins, deriving from different parts in the male reproductive tract and of different molecular weight, affect male competitive fertilization success in C. maculatus. Our results highlight the diverse effects of seminal fluid proteins and show that the function of such proteins can be contingent upon female mating status. We also document effects of different size fractions on female mating receptivity and egg laying rates, which can serve as a basis for future efforts to identify the molecular identity of seminal fluid proteins and their function in this model species.     

Spermatozoa production in male <Emphasis Type="Italic">Varroa destructor</Emphasis> and its impact on reproduction in worker brood of <Emphasis Type="Italic">Apis mellifera</Emphasis>

Reproduction in Varroa destructor exclusively takes place within the sealed honey bee brood cell and is, therefore, limited by the duration of the postcapping period. Oogenesis, ontogenetic development and mating must be optimized to ensure the production of as many mated daughter mites as possible. One adult male mite has to mate with up to five sister mites and transfer 30–40 spermatozoa to each female. We analyzed the production and transfer of male spermatozoa during a reproductive cycle by counting all spermatozoa in the genital tracts of the male and daughter mites in 80 worker brood cells at defined times after cell capping. We could show that spermatozoa production in male mites is an ongoing process throughout their adult lifetime starting after the adult molt. The spermatozoa are transferred to the females in an early non-capacitated stage and require further maturation within the female’s genital tract. Our study points out that a Varroa male has at any time in the brood cell enough spermatozoa to inseminate all daughter mites but does not waste energy in producing a big surplus. In total one male produced, on average, 125 spermatozoa during a reproductive cycle in worker brood which is sufficient for successful matings with at least three daughter mites. Spermiogenesis in Varroa males represents therefore a further adaptation to the limited time available for reproduction.     

Divergence in genital morphology may contribute to mechanical reproductive isolation in a millipede

Genitalia appear to evolve rapidly and divergently in taxa with internal fertilization. The current consensus is that intense directional sexual selection drives the rapid evolution of genitalia. Recent research on the millipede Antichiropus variabilis suggests that the male genitalia are currently experiencing stabilizing selection – a pattern of selection expected for lock‐and‐key structures that enforce mate recognition and reproductive isolation. Here, we investigate how divergence in genital morphology affects reproductive compatibility among isolated populations of A. variabilis. Females from a focal population were mated first to a male from their own population and, second, to a male from one of two populations with divergent genital morphology. We observed variation in mating behavior that might indicate the emergence of precopulatory reproductive barriers: males from one divergent population took significantly longer to recognize females and exhibited mechanical difficulty in genital insertion. Moreover, we observed very low paternity success for extra‐population males who were successful in copulating. Our data suggest that divergence in genital shape may be contributing to reproductive isolation, and incipient speciation among isolated populations of A. variabilis.     

Sperm transport and storage in the agile antechinus (Antechinus agilis).

This study was an examination of the timing of ejaculation and the dynamics of sperm transport in the female reproductive tract of the agile Antechinus (Antechinus agilis) and the relationship of these parameters to single and multiple matings. Mating in this species is characteristically long compared with that of other mammals, lasting for up to 8-12 h during which time the pair remain locked together. After the first hour of mating, only approximately 40% of males had ejaculated, but by the third hour all males had ejaculated. The total number of spermatozoa extracted from the female tract remained at approximately 30 x 10(3) spermatozoa per side over the next 9 h of copulation. After completion of male/female access (12 h), approximately 56% of spermatozoa extracted were located in the lower isthmic region of the oviduct where specialized sperm storage crypts are located. The number of spermatozoa extracted from the female reproductive tract did not decline over the next 3 days, but there was a change in the distribution of spermatozoa with an increase in the proportion of extracted spermatozoa stored in the lower isthmus (approximately 76%). However, 7 to 14 days after mating, only approximately 30% of the stored spermatozoa ( approximately 9.4 x 10(3) spermatozoa per side) were still present in the isthmus. When females were mated with a second male on a consecutive day, the sperm numbers extracted from the tract were about twice that deposited during single mating, with sperm transport to the lower isthmus occurring over a similar time frame. Although the occurrence of extended copulations in the wild has not yet been confirmed, these laboratory results suggest that similar periods of copulation are likely, since completion of the ejaculation process requires at least 3 h. The extended copulation in A. agilis reduces the possibility of an early second mating, which might interfere with the normal transport and crypt colonization of spermatozoa through competition.     

Morphology and histology of male and female reproductive systems in the inseminating species Scoloplax distolothrix (Ostariophysi: Siluriformes: Scoloplacidae)

The morphology and histology of male and female reproductive systems were examined in Scoloplax distolothrix. Internal insemination was documented in this species by the presence of sperm within the ovaries. Mature males and females have elongated genital papillae, exhibiting a tubular shape in males and a plain heart‐shape with two median protuberances in females. The testes are two elongated structures that converge ventrally, under the intestine, towards the genital papilla. They are joined at the caudal end, forming an ovoid single chamber for sperm storage. Secretory regions were not observed. In the lumen of the testicular tubules, spermatozoa can be tightly packed along their lengths, but do not constitute a spermatozeugmata. The lumen of the sperm storage chamber and spermatic duct are filled with free spermatozoa without the accompanying secretions. The ovaries are bird‐wing shaped, saccular structures that converge ventrally under the intestine, towards the genital papilla. They are joined at the caudal end, forming a tubular chamber possibly destined for oocyte storage. An oviduct with an irregular outline connects the chamber to the tubular region of the genital papilla. No distinct sperm storage structure was found in the ovaries. The unique male and female genital papillae suggest that these structures are associated with the reproductive mode in scoloplacids, representing evidence for insemination. The occurrence of free spermatozoa, without the accompanying secretions and not arranged in a spermatozeugmata can be associated with the presence of a tubular male genital papilla for sperm transfer to the female genital tract. This reinforces the idea that sperm packets are not necessary for all inseminating species. The male reproductive system in scoloplacids is very different from that in auchenipterids, a second catfish family with insemination, which indicates that the occurrence of insemination is not connected to the internal morphology of reproductive organs. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

Sperm release behaviour and fertilization in the grass goby

Nesting males of the grass goby Zosterisessor ophiocephalus showed bouts, with intervals of c. 30 min duration, of upside-down movements while rubbing its genital papilla onto the ceiling of its burrow. Such behaviour was shown during female courting and spawning, and even after female removal. Observations showed that this behaviour was associated with the release of a sperm trail on the substratum and clumped spermatozoa in water, agglutinated with a mercury-bromophenol blue and PAS positive glycoprotein secretion of the sperm duct glands. Agglutination in the secretion delayed the activation of spermatozoa and contributed a steady supply, for up to 40 min, of motile spermatozoa during prolonged egg laying by the female. Sperm released before egg laying achieved c. 50% fertilization success compared with nearly 100% obtained if the sperm was released during egg laying. The sperm release behaviour may improve the nest owner's reproductive success against intruders or sneakers. It also allows defence of the nest while the female is spawning and may allow the male to court other females in the proximity.     

Prolonged receptivity to the male and the fate of spermatozoa in the female black mastiff bat, Molossus ater

Observations were made on the reproductive biology of black mastiff bats maintained in a laboratory colony. Many of the females were inseminated within 24 h after the introduction of the males, and most exhibited a period of 10-50 days during which spermatozoa were present in their vaginal smears almost every day. The frequency of sperm-positive smears began to fall off around the time of implantation, but some smears taken much later in pregnancy were positive. The extent to which spermatozoa in the smears came from reservoirs in the female tract could not be thoroughly investigated, but evidence was obtained that the females have more than a limited period of oestrus. Female courtship behaviour and new copulations were sometimes observed many days after the start of the breeding activity. Also, histological studies of the reproductive tracts of females which had recently mated revealed that many were not in a periovulatory condition. Intact spermatozoa were usually found in the uterine horns and distal oviducts of preovulatory bats and those carrying tubal ova. Spermatozoa were absent from the oviducts of animals bearing early uterine embryos, and were much less abundant in the uterine horns after the start of implantation. Many of the excess spermatozoa appeared to have been expelled into the upper cervix where phagocytic leucocytes were commonly observed in the lumen. Some sperm components were also taken up by epithelial cells in the oviducts and uterine horns.     

Copulation patterns in the golden orb-web spider Nephila madagascariensis

A consequence of multiple mating by females can be that the sperm of two or more males directly compete for the fertilisation of ova inside the female reproductive tract. Selection through sperm-competition favours males that protect their sperm against that of rivals and strategically allocate their sperm, e.g., according to the mating status of the female and the morphology of the spermatheca. In the majority of spiders, we encounter the otherwise unusual situation that females possess two independent insemination ducts, both ending in their own sperm storage organ, the spermatheca. Males have paired mating organs, but generally can only fill one spermatheca at a time. We investigated whether males of the African golden orb-web spider Nephila madagascariensis can prevent rival males from mating into the same spermatheca and whether the mating status of the female and/or the spermatheca causes differences in male mating behaviour. There was no significant difference in the duration of copulations into unused spermathecae of virgin and mated females. We found that copulations into previously inseminated spermathecae were generally possible, but shorter than copulations into the unused side of mated females or with virgins. Thus, male N. madagascariensis may have an advantage when they mate with virgins, but cannot prevent future males from mating. However, in rare instances, parts of the male genitals can completely obstruct a female genital opening.     

Copulation, genital damage and early death in Callosobruchus maculatus

Antagonistic sexual coevolution stems from the notion that male and female interests over reproduction are in conflict. Such conflicts appear to be particularly obvious when male genital armature inflicts damage to the female reproductive tract resulting in reduced female longevity. However, studies of mating frequency, genital damage and female longevity are difficult to interpret because females not only sustain more genital damage, but also receive more seminal fluid when they engage in multiple copulations. Here, we attempt to disentangle the effects of genital damage and seminal fluid transfer on female longevity in the beetle Callosobruchus maculatus (Coleoptera: Bruchidae). Males copulating for the sixth time in succession inflicted greater levels of genital damage, but transferred smaller ejaculates in comparison with virgin males. The number of copulations performed by males was negatively related to female fecundity and positively related to female longevity, suggesting a trade-off between fecundity and longevity. However, inclusion of fecundity as a covariate revealed sperm and/or seminal fluid transfer to have a negative impact on female longevity above that caused by the fecundity-longevity trade-off. The consequences of multiple copulations on female longevity were examined. Females that mated twice laid more eggs and died sooner than those that mated once. However, incorporation of fecundity as a covariate into our statistical model removed the effect of female mating frequency on female longevity, indicating that double-mated females suffer greater mortality owing to the trade-off between fecundity and longevity. Males of this species are known to transfer very large ejaculates (up to 8% of their body weight), which may represent a significant nutritional benefit to females. However, the receipt of large ejaculates appears to carry costs. Thus, the interpretation of multiple mating experiments on female longevity and associated functional explanations of polyandry in this species are likely to be complex.     

Immunogenetic studies of spontaneous abortion in mice. Preimmunization of females with allogeneic cells

CBA females (H-2k) mated with DBA2 males (H-2d) exhibit a high rate of fetal resorption (30%) when compared with the CBA female BALB/c male, CBA female/CBA male, DBA2 female/CBA male, DBA2 female/DBA2 male combinations (6 to 8%). Preimmunization of CBA females with spleen cells from DBA2, BALB/c, or CBA males were performed in order to test their effects on CBA maternal tolerance of (CBA X DBA2)F1 fetuses. Only preimmunization with BALB/c male cells was effective in decreasing resorption; cells from BALB/c females had no effect. In order to further test 1) the role of non-MHC-encoded antigens present in the BALB/c male background, 2) the necessity of an additional H-2 difference, and 3) whether or not the phenomenon is H-2d restricted, preimmunizations were performed by using cells from congenic BALB/k (H-2k), BALB/b (H-2b), or BALB/c (H-2d). Only the latter treatment was efficient, which suggests that the paternal H-2d haplotype must be presented in synergy with some non-MHC-encoded antigens in the BALB/c male background. Immunogenetic studies with cells from nine recombinant inbred strains that reassorted DBA2 and BALB/c genomes showed that three of them behave like BALB/c and six like DBA2. This would suggest that the genetic determinism of this phenomenon is simple.     

Spermatogenesis, changes in reproductive structures, and time constraint associated with insemination in Podisus nigrispinus

Males of the predatory stinkbug, Podisus nigrispinus (Dallas) (Hemiptera: Pentatomidae), accomplish long and multiple matings. We hypothesize that this behavior is due to time requirement for spermatozoa production and their transference to the females. Thus, this work investigated the effect of mating status of males and mating duration on spermatozoa transference to the females and the location of spermatozoa in the male reproductive tract during mating. On females, morphological alterations on female spermatheca and associated structures during a mating were investigated. Analyses of male reproductive tract showed presence of spermatozoa in the lumen of vas deferens was independent of mating status (ca. virgin, 0, 12 and 24 h after having a full mating), indicating continuous spermatogenesis which is supported by the absence of a seminal vesicle for spermatozoa storage. Female spermatheca had no changes associated with the duration of mating. However, females exhibited spermathecal elastic duct swelling by 30 min of mating duration. The success of males in filling the female spermatheca with spermatozoa depends on duration of mating. Thus, the results indicate that multiple mating is a requirement for reproductive success in the species by transference of spermatozoa and accessory substances stored in the female spermathecal duct. Likewise, the long mating is a male requirement to transfer materials in appropriate amount to the female but it is not dependent on spermatozoa alone.     

The role of male age, sperm age and mating history on fecundity and fertilization success in the hide beetle

Models of age-related mate choice predict female preference for older males as they have proven survival ability. However, these models rarely address differences in sperm age and male mating history when evaluating the potential benefits to females from older partners. We used a novel experimental design to assess simultaneously the relative importance of these three parameters in the hide beetle, Dermestes maculatus. In a two-part experiment we first explored age-related male mating success and subsequently examined the consequences of male age, sperm age and male mating history on female fecundity and fertilization success. In a competitive mating environment, intermediate-age males gained significantly higher mating success than younger or older males. To test the consequences for females of aged-related male mating success, a second set of females were mated to males varying in age (young, intermediate-age and old), in numbers of matings and in timing of the most recent mating. We found that male age had a significant impact on female fecundity and fertilization success. Females mated to intermediate-age males laid more eggs and attained consistently higher levels of fertilization success than females with young and old mates. A male's previous mating history determined his current reproductive effort; virgin males spent longer in copula than males with prior mating opportunities. However, differences in copulation duration did not translate into increased fecundity or fertilization success. There was also little evidence to suggest that fertilization success was dependent on the age of a male's sperm. The experiment highlights the potential direct benefits accrued by females through mating with particular aged males. Such benefits are largely ignored by traditional viability models of age-related male mating success.     

Effect of mating on sperm distribution in the reproductive tract of the male rat

Extragonadal sperm reserves in male rats were measured in different regions of the genital tract before and subsequent to normal ejaculation. In sexually rested rats, the sperm count (million spermatozoa for the paired organs) in different regions was: distal vas, 18; proximal vas, 9.8; cauda epididymidis, 229; caput + corpus epididymidis, 154. Following mating, the sperm count was reduced in the proximal and distal vas deferens and in the cauda epididymidis. The reproductive tract of mated females was found to contain 29% (no copulatory plug) or 59% (with copulatory plug) of the estimated mean ejaculate, which was estimated from the difference between the sperm counts in the sexually rested rat and following ejaculation. It is concluded that in the rat the immediate source of spermatozoa for ejaculation is the cauda epididymidis, with a smaller contribution arising from the vas deferens.     

Mating strategy in Aleochara bilineata: sperm storage and allocation

Abstract By contrast to females that can maximize reproductive success with only one or a few copulations, males generally increase their fitness with frequency of mating. Sperm storage and allocation is therefore crucial for both male and female fitness. Sperm storage in Aleochara bilineata (Coleoptera; Staphylinidae) is investigated by measuring the number of spermatozoa stored in the female spermatheca after single, double or triple successive copulations with different males. The potential advantages of polyandry are studied in terms of the number of sperm stored by females mated twice with the same male (i.e. repeated copulation), compared with females mated twice with two different virgin males (i.e. polyandry). Level of polygyny is also estimated by measuring sperm allocation when ten successive mates are offered to a virgin male. Aleochara bilineata females store the sperm of the same or different males additively, suggesting no advantage for polyandry in terms of the number of sperm stored. A virgin male is able to inseminate ten different females but the number of sperm transferred decreases linearly. Finally, the latencies and durations of copulations are measured in all experiments to estimate changes according to the male or female status (i.e. virgin or mated). The latency before mating is higher when females are virgin than when females have already mated.     

Prostaglandin and prostaglandin synthetase in the cricket, Acheta domesticus

Prostaglandin (PG) synthetase was present in the testes, seminal vesicles, and spermatophores of the male house cricket, Acheta domesticus. The enzyme was not detected in bursa copulatrix, spermatheca, spermathecal canal, and oviducts from virgin females, while substantial activity was measured in the same tissue from mated females. The female appears to receive the enzyme from the spermatophore. A PGE₂-like material was detected by radioimmunoassay in A. domesticus testes and to a lesser extent in the remainder of the male reproductive tract. PG went undetected in virgin female reproductive tissues, while the same tissues from mated females contained an average of 589 pg of PGE₂-like material per female. In in vivo studies, injected PGE₁, PGE₂, and to a smaller degree PGF_2α stimulated oviposition by virgin females. Moreover, N-acetyl-p-aminophenol, a PG synthetase inhibitor, suppressed oviposition in mated females. Post-copulatory PG biosynthesis in the female reproductive tract might be partially responsible for triggering oviposition in A. domesticus. Since PG synthetase appears to be acquired from the male, it could be considered a primer pheromone.