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In addition to the stimulatory e ffe c ts o f primer pheromones described in section 1.2.1.2., there are many examples o f pheromonally-induced suppression o f reproduction in rodents. These may act in d iffe re n t ways to cause inhibitin o f fe rtility , fo r example by delaying sexual maturation and puberty, by suppression o f oestrus and ovulation, and by interfering with embryo implantation.

Perhaps the most commonly reported form o f pheromonal contraception observed in rodents is suppression o f puberty. First described by van der Lee St Boot (1955), to explain the delay in sexual maturation and increase in cycle length seen in group-housed female mice, the e ffe c t is now known to be due to a chemosignal present in the voided urine o f group housed females (Cowley St Wise, 1972). Interestingly, the active puberty delaying fa c to r is present in the bladder urine of all female mice, irrespective o f age or density o f grouping, suggesting that activation and deactivation o f the pheromone may occur in the urethra. This was confirmed when bladder urine containing the active pheromone was incubated with homogenised urethras from singly housed females, and the puberty delaying ability lost (McIntosh St Drickamer, 1977). This unusual pheromonal control system is itself under the influence o f yet another chemosignal, a fa c t that became apparent a fte r group-housed females had their vomeronasal organs surgically removed. These individuals failed to release the puberty delay pheromone because an essential o lfa cto ry signal was prevented from acting (Lepri et al, 1985). Further evidence to support this was obtained when singly-caged female mice were induced to release the puberty delay chemosignal a fte r exposure to urine from group housed females (Drickamer, 1982). Therefore, when female mice are housed in groups a chemosignal is produced which, acting via the vomeronasal organ causes the deactivation mechanism o f the puberty delay pheromone in the urethra to be "switched o ff" and the active puberty delay pheromone is excreted in the voided urine (Drickamer, 1982; Lepri et al, 1984). This is perhaps the best researched example o f pheromonally-induced suppression o f reproduction in rodents, and a diagrammatic representation o f the sequence o f events is summarised in Figure 1.3.

FIGURE 1.3 - Control and release o f the mouse puberty delay pheromone. VNO= Vomeronasal organ, A= active puberty delay signal, a = inactive puberty

delay signal. GROUP HOUSED

W

The presence o f the puberty delay pheromone in mouse urine is not a ffe c te d by ovariectomy, but appears to be associated with adrenal function, because it is abolished by adrenalectomy (Drickamer et al, 1978; Drickamer & McIntosh, 1980). It is also potent and e ffe c tiv e a t very low doses. Drickamer Cl 984) has shown that a daily dose o f 0.001 ml urine from grouped females produced a significant delay in puberty when applied to the noses o f juvenile females, compared to controls, while doses o f 0.0001 ml were still e ffe c tiv e but the response was reduced.

The mouse puberty delay pheromone is one o f the few mammalian primer pheromones to have been chemically characterised. Analysis o f urine using gas chromatography-mass spectrometry techniques has shown th a t the pheromone is not one, but several volatile compounds. Comparing analytical data from intact, group-housed female mouse urine which contains the pheromone, to that o f urine from adrenalectomized females which do$s not contain the pheromone, differences in concentrations o f certain volatile compounds were seen. In particular, six compounds were consistently higher in urine containing the pheromone: 2-heptanone, trar»s-4-hepten-2-one, tr a n s -5 - hepten-2-one, n-pentyl acetate, c/s-2 -p e n te n -1-y l-ac e ta te and 2,5-dimethyl pyrazine. When these compounds were all collectively spiked into the urine o f adrenalectomized mice and this urine bioassayed, they were found to have the full biological activity o f the pheromone. The pyrazine also had some e ffe c t when added on its own to adrenalectomized mouse urine, significantly increasing the time to fir s t oestrus compared with controls. The two acetate esters also significantly delayed puberty compared to controls, but the three ketones on their own had no significant e ffe c t. These results illustrate that compounds may act synergistically to produce the final pheromonal e ffe c t (Novotny et al, 1986).

The influence o f suppressing pheromones produced by group-housed female mice can extend beyond puberty delay in juveniles to cause inhibition o f oestrus and ovulation in mature animals. Elevation o f prolactin is implicated in the physiological mechanism mediating the response (Reynolds & Keverne, 1979; fo r review see Milligan, 1980). In the mouse, prolactin is a luteotrophic agent and therefore in cycling females its elevation will maintain the corpus luteum and its secretion o f progesterone. This results in extended periods o f dioestrus (the luteal phase o f the cycle) similar to those seen during pseudopregnancy, with a delay in the ensuing oestrus and ovulation. In large groups (30 animals) complete suppression o f ovulation may occur

resulting in a state of anoestrus (Whitten, 1959), presumably due to extreme or sustained hyperprolactinaemia.

The suppressing pheromone released from group-housed female mice has also been been found in the urine o f wild house mice. Urine collected from females living in high density populations was able to suppress puberty in captive singly-housed juvenile females. However, urine collected when population density was low failed to delay puberty (Massey and Vandenbergh, 1980; Coppola, 1986). This density-dependent release o f pheromones may therefore play an important role in regulating natural rodent populations by controlling the rate of sexual maturation.

Suppression o f puberty has also been observed in dense laboratory populations o f the prairie deer mouse, Peromyscus maniculatus bairdii where only about 10% o f females reproduce even in conditions o f excess food and water. The non-breeding or suppressed individuals have reduced body weights and underdeveloped reproductive tra c ts (Kirkland & Bradley, 1986). However, when these non-breeders are removed from the group they become sexually active and 70% reproduce within 50 days (Terman, 1973). The specific nature o f the environmental cues responsible fo r this suppression remains unclear, although both behavioural or ta c tile cues, and o lfacto ry chemosignals have been implicated (Terman, 1987). In this species, male deer mice are also inhibited in their sexual maturation - juveniles exposed to soiled bedding containing urinary chemosignals from the cages o f adult males, had growth o f testes and seminal vesicles retarded. The urine o f adult females had no e ffe c t on males, implying a specific pheromone was involved in male suppression (Lawton & Whitsett, 1979). However, other studies o f juvenile male prairie deermice have not found such clear evidence fo r the involvement of pheromones in suppression o f reproduction (Terman, 1987). Kirkland & Bradley (1986) have shown th at serum prolactin levels are reduced in both reproductively inhibited males and females, suggesting that suppression of prolactin may be involved in the physiological suppression mechanism. In the ra t, hypoprolactinaemia has been shown to be associated with delay puberty in females (Advis et al, 1981).

Pheromonal inhibition o f puberty is a common fe a tu re among high- density populations o f microtine rodents, e.g. bank voles , Clethrionomys glareolus (Kruczek and Marchlewska-Koj, 1986); pine voles, Microtus pinetorum (Lepri, 1986; Lepri & Vandenbergh, 1986); Californian voles, M. californicus and the prairie vole, M. ochrogaster (Batzli et al, 1977). However, puberty

d e l a y is n o t n o r m a l l y s e e n in t h e m e a d o w v o l e M. p e n n s y l v a n i c u s a n d t o e x p l a i n t h i s s p e c i e s d i f f e r e n c e B a t z l i e t a l h y p o t h e s i s e d t h a t r e p r o d u c t i v e s u p p r e s s i o n m a y be r e l a t e d t o h a b i t a t . T h e y a r g u e d t h a t in t h e c a s e o f M. c a h f o r n i c u s a n d M. o c h r o g a s t e r , w h i c h l i v e in e x t e n s i v e g r a s s l a n d h a b i t a t s , t h e b e s t s u r v i v a l s t r a t e g y w o u l d b e f o r t h e y o u n g t o l e a v e t h e f a m i l y b e f o r e b r e e d i n g . R e p r o d u c t i v e s u p p r e s s i o n w o u ld e n f o r c e t h i s . C o n v e r s e l y , t h e m e a d o w v o l e s h a b i t a t is s m a l l p a t c h e s o f m o i s t m e a d o w o r m a r s h a n d n e w a r e a s m u s t c o n t i n u o u s l y b e i n v a d e d . B r e e d i n g a m o n g s t s i b l i n g s w o u l d b e f a v o u r e d b e c a u s e s t r a n g e m a t e s m a y n o t a l w a y s b e a v a i l a b l e a n d r e p r o d u c t i v e s u p p r e s s i o n w o u l d b e a d i s a d v a n t a g e . F u r t h e r e v i d e n c e in s u p p o r t o f t h i s t h e o r y w a s o b t a i n e d w h e n t w o o t h e r s p e c i e s o f m i c r o t i n e r o d e n t s w e r e s t u d i e d . T h e b r o w n le m m in g , L e m m u s s i b i r i c u s i n h a b i t s e x t e n s i v e r e g i o n s o f a r c t i c t u n d r a , a n d e x h i b i t s r e p r o d u c t i o n s u p p r e s s i o n , w h i l e t h e t u n d r a v o t e , M. o e c o n o m u s is l i m i t e d t o s m a l l p a t c h e s o f h a b i t a t w i t h i n t h e t u n d r a , a n d l i k e t h e m e a d o w v o l e , d o e s n o t h a v e r e p r o d u c t i v e s u p p r e s s i o n ( F a c e m i r e & B a t z l i , 1 9 8 3 ) . R e p o r t s o f r e p r o d u c t i v e s u p p r e s s i o n w h i c h a r e m a n i f e s t a s a p o s t - c o p u l a t o r y b l o c k a r e s c a r c e , t h e b e s t k n o w n e x a m p l e b e i n g t h e m a l e - i n d u c e d B r u c e e f f e c t o b s e r v e d in f e m a l e m i c e ( B r u c e , 1 9 5 9 ) . T h e r e i s s t i l l s o m e d e b a t e a s t o w h e t h e r o r n o t t h i s p h e n o m e n o n is a c o m m o n o c c u r r e n c e in w i l d p o p u l a t i o n s , a n d m a y be a l a b o r a t o r y a r t i f a c t ( K e v e r n e & R o s s e r , 1 9 8 6 ) . H o w e v e r , H a i g h e t a l ( 1 9 8 8 ) h a v e r e p o r t e d a n e x a m p l e o f p h e r o m o n a l l y - i n d u c e d i m p l a n t a t i o n f a i l u r e in t h e w h i t e - f o o t e d m o u s e , P e r o m y s c u s le u c o p u s , a c l o s e r e l a t i v e o f t h e p r a i r i e d e e r m o u s e . T h e y o b s e r v e d t h a t in c a p t i v i t y , f e m a l e s f a i l e d t o p r o d u c e o f f s p r i n g w h e n p a i r e d w i t h a m a l e f r o m w e a n i n g t o 1 5 0 d a y s o f a g e i f a n a d u l t f e m a l e o r h e r o d o u r w a s p r e s e n t . T h e s a m e r e s u l t s w e r e a l s o o b t a i n e d in a n o t h e r e x p e r i m e n t l a s t i n g 3 0 0 d a y s . T h i s is w e ll i n e x c e s s o f t h e n o r m a l l i f e e x p e c t a n c y o f t h e w h i t e - f o o t e d m o u s e in t h e w il d a n d t h e r e f o r e r e p r e s e n t s a c o m p l e t e s u p p r e s s i o n o f r e p r o d u c t i o n . T h e s e s u p p r e s s e d f e m a l e s w e r e f o u n d t o b e m a t i n g a n d o v u l a t i n g , w i t h f e r t i l i z a t i o n o c c u r r i n g . S o i t w o u ld a p p e a r t h a t t h e b l o c k t o r e p r o d u c t i o n