*5.2. Community Engagement*

There is a growing recognition that citizens should be actively involved as key stakeholders in the watershed planning process. By engaging communities, policymakers can gain local knowledge, increase fairness and equity in decision making, and avoid failure due to non-acceptance [7]. An effective EDSS can allow citizens to take a participatory approach and highlight the various advantages and disadvantages of different scenarios [27].


**5.2.2 NA metabolism**

104 Sexual Dysfunctions – Special Issue

has been reduced in groups with prenatal exposure of methylbenactyzine and ganglerone. Ratio indexes 5-HIAA/5-HT have been increased only in those groups in which decrease of

**Control** 0,244±0,018 0,976±0,068 0,637±0,037 0,608±0,035

**G-10** 0,228±0,026 1,120±0,061 0,470±0,023\* 0,578±0,041

**G-13** 0,194±0,022 1,192±0,078\* 0,461±0,030\* 0,636±0,028

**G-18** 0,340±0,020\* 1,134±0,062\* 0,540±0,026 0,587±0,032

**M-10** 0,344±0,022\* 1,240±0,059\* 0,378±0,033\* 0,538±0,039

**M-18** 0,402±0,031\* 1,188±0,069\* 0,490±0,041\* 0,550±0,039

Table 3. Turnover of DA and 5-HT neurotransmitters in hippocampus and hypothalamus at 2-month-old rat offspring exposed to methylbenactyzine or ganglerone at different periods

Research of neurotransmitters turnover in the hippocampus has shown that rats offspring subjected to exposure of cholinolytics have an enchancement of the turnover of the basic mediators, leading to decrease of concentration of these neurotransmitters in the hippocampus. Noted increase of DA turnover in the hippocampus of rats offspring prenatally subjected to exposure by methylbenactyzine (tab. 3) and significant increase of 5- HT turnover in the hippocampus of the offsprings, subjected to prenatal exposure both

The neurochemical status of DA in the hypothalamus of 2-month-old rats offsprings subjected to prenatal exposure of cholinolytics, was characterised by significant decrease of dopaminergic activity in relation to control (fig. 5). In comparison with the methylbenactyzine, prenatal exposure of the ganglerone, caused more appreciable remote

Concentration of the DA has been reduced in all groups - G10 - G18 (31,7 % - 36,9 %, р≤0,001) with maximally low value in G13 group. Among offsprings with prenatal exposure to methylbenactyzine significant decrease of DA noted only in M18 group (17,6 %, р≤0,05). Dynamics of DOPAC change in relation to control group was similar to dynamics of DA

significant 1,5 - 2 times decrease of DOPAC concentration in all groups was noted.

of prenatal development. (M ± m) \*-p < 0.05 compared with the control group

HIPPOCAMPUS HYPOTHALAMUS

**DA 5-HT DA 5-HT**

mediator level became perceptible.

methylbenactyzine and ganglerone.

changes of DA level in the hypothalamus of these offsprings.

**5.2 Hypothalamus 5.2.1 DA metabolism**

groups

The neurochemical status of NA in the hypothalamus was characterised by rising of processes of degradation of a mediator without enchancement of synthesis processes. In all studied groups both males and females had a decreased NA level and high MHPG content in the hypothalamus in comparison with control offsprings' grou. In comparison with methylbenactyzine, NA concentration in groups with prenatal exposure to ganglerone, has been reduced more considerably (in a greater degree in G10 group - decrease of the NA of 26,2 %, р≤0,001). The content of MHPG metabolite, on the contrary, has been raised in all groups in 1,5 - 2,5 times.

Fig. 5. Contents of dopamine (DA), noradrenaline (NA), and serotonin (5-HT) (ng/mg of wet tissue) in the hypothalamus in two-month-old rat offspring exposed to methylbenactyzine or ganglerone at different periods of prenatal development. \*p < 0.05 compared with the control group. For further details see caption to Fig. 1.

#### **5.2.3 5-HT metabolism**

Dynamics of 5-HT change in the hypothalamus was similar to other mediators – rats offsprings from G10 - G18 groups, where mediator level has been reduced in significant limens (accordingly, 24,3 % - 35,4 %, р≤0,001), had more radical changes in 5-HT content in G10 - G18 groups. In methylbenactyzine groups 5-HT concentration in the hypothalamus strengthened (in M10 group on 14,2 %, р≤0,05). 5-HIAA metabolite content in the hypothalamus of all the studied groups was similar to dynamics of the mediator.

The greatest changes of the neurotransmitters turnover in the hypothalamus have been detected concerning a DA both of rats males and females (tab. 1). DA turnover has considerably reduced in the males hypothalamus in all experimental groups in comparison with control group. The ratio index 5-HIAA/5-HT was more stable, except for G10 group of females which index was reduced. In the same group increase of 5-HT level at the stable content 5-HIAA was noticed that shows enchancement of serotoninergic synaptic activity in

Development of Male Sexual Function After Prenatal Modulation of Cholinergic System 107

within limbic system. Long-term effects of prenatal exposures of cholinolytics within amygdala nuclei have been brightly expressed. Study's results show significant decrease of DA, 5-HT and NA mediators content in the amygdala of the males prenatally subjected to exposure of gangleron, larger degree on 10-13 days of gestation (fig. 7). Prenatal exposure of M-cholinolytic methylbenactyzine had no strongly pronounced consequences on

development of neurotransmitter systems in the amygdala

**Control**

postnatal life.

\*

**М-13**

**М-18**

control group. For further details see caption to Fig. 1.

**Control**

**0,00**

**0,05**

**0,10**

**0,15**

**0,20**

**0,25**

**G-10**

**G-13**

Fig. 7. Contents of dopamine (DA), noradrenaline (NA), and serotonin (5-HT) (ng/mg of wet tissue) in the amygdala in two-month-old rat offspring exposed to methylbenactyzine or ganglerone at different periods of prenatal development. \*p < 0.05 compared with the

Changes of the neurotrasmitter status in the amygdala can affect functioning of other brain structures participating in regulation of sexual function, by means of neuronal connection of this structure with hippocampus and hypothalamus. It is shown that destruction of hypothalamo-amigdaloid connection, damage or irritation of the amygdala lead to neurohumoral alterations and sexual behavior abnormalities (Akmaev, 1993; Swanson, 1998; Dominguez, 2001). It is not excluded that optimization of integrative connections within limbic system and hypothalamus is extremely important concerning the effects of

Results of these researches show that pregnant females in "critical periods" of the embryo prenatal development exposure to M- and N-cholinoblockers can cause long-term changes in neurotransmitter systems activity in investigated structures of the rats brain in their

Exposure of M- and N-cholinolytics in the prenatal period leads to significant decrease of dopaminergic activity in the hippocampus and hypothalamus of rats offspring in comparison with control group. Results of various researches prove that defects of DA synaptic activity in the hippocampus at offspring can accompany to hippocampusassociated behavioral deficiencies at puberal individuals (Yanai, 1984; Smith, et al. 1986;

\* \*

**G-18**

**NA**

**М-10**

\*

\*

**М-13**

**М-18**

**Control**

**0,0**

**0,2**

**0,4**

**0,6**

**0,8**

**1,0**

**G-10**

**G-13**

\*

\*

**G-18**

\* \*

**5-HТ**

**М-10**

**М-13**

**\* -p<0,05**

**М-18**

\* \*

the hypothalamus. NA turnover in the hypothalamus of experimental groups was comparable with the data of control group though the mediator content and its metabolite has been reduced in all groups.

Sexual dimorphism in effects of prenatal exposure of M- and N-cholinolytics on dopaminergic system (fig.6) has been detected. In case of males offspring that were exposed to prenatal exposure of a ganglerone N-cholinolitics the decrease of DA concentration in the brain structures was noted; in case of rats females – the same reduction after prenatal exposure to M-cholinolytic methylbenactyzine.

Fig. 6. Sexual dimorphism according to dophamine concentration (ng/mg of wet tissue) in the hippocampus in two-month-old rat offspring exposed to methylbenactyzine or ganglerone at different periods of prenatal development. \*p<0.05 compared with the control group. Notations: - **♂, - ♀ -** simbols of the genetical sex of offspring, accordingly males and females.

#### **5.3 Amygdala**

For realization of adaptive and sexual behavior dopaminergic system of amygdala is also important. The amygdala initiates the organization of adequate behavior to the situation and by means of influence on the hypothalamus and vegetative excitatory system frames conforming hormonal and neurovegetative assurance to this behavior (Simonov, 1987). According to many researchers an amygdaloid complex is responsible for integration of emotional expressions, characteristic for sexual motivation (Newman, 1999; Dominguez, 2001).

Dynamics of neurotransmitters level in the amygdala was similar to the neurochemical status in the hippocampus that once again confirms their morphophysiologycal generality

the hypothalamus. NA turnover in the hypothalamus of experimental groups was comparable with the data of control group though the mediator content and its metabolite

Sexual dimorphism in effects of prenatal exposure of M- and N-cholinolytics on dopaminergic system (fig.6) has been detected. In case of males offspring that were exposed to prenatal exposure of a ganglerone N-cholinolitics the decrease of DA concentration in the brain structures was noted; in case of rats females – the same reduction after prenatal

has been reduced in all groups.

exposure to M-cholinolytic methylbenactyzine.

**G-10 G-13 G-18**

\*

**Control**

**0,0**

females.

2001).

**5.3 Amygdala**

**1,0**

**2,0**

**3,0**

**4,0**

**5,0**

**М-10**

**DA - ♂**

\* \*

**М-18**

**Control**

**0,0**

Fig. 6. Sexual dimorphism according to dophamine concentration (ng/mg of wet tissue) in

ganglerone at different periods of prenatal development. \*p<0.05 compared with the control group. Notations: - **♂, - ♀ -** simbols of the genetical sex of offspring, accordingly males and

For realization of adaptive and sexual behavior dopaminergic system of amygdala is also important. The amygdala initiates the organization of adequate behavior to the situation and by means of influence on the hypothalamus and vegetative excitatory system frames conforming hormonal and neurovegetative assurance to this behavior (Simonov, 1987). According to many researchers an amygdaloid complex is responsible for integration of emotional expressions, characteristic for sexual motivation (Newman, 1999; Dominguez,

Dynamics of neurotransmitters level in the amygdala was similar to the neurochemical status in the hippocampus that once again confirms their morphophysiologycal generality

the hippocampus in two-month-old rat offspring exposed to methylbenactyzine or

**0,5**

**1,0**

**1,5**

**2,0**

**2,5**

**3,0**

**3,5**

**4,0**

**G-10**

**G-13**

**G-18**

**DA-♀**

**М-10**

**М-18**

\*

\*

\*

within limbic system. Long-term effects of prenatal exposures of cholinolytics within amygdala nuclei have been brightly expressed. Study's results show significant decrease of DA, 5-HT and NA mediators content in the amygdala of the males prenatally subjected to exposure of gangleron, larger degree on 10-13 days of gestation (fig. 7). Prenatal exposure of M-cholinolytic methylbenactyzine had no strongly pronounced consequences on development of neurotransmitter systems in the amygdala

Fig. 7. Contents of dopamine (DA), noradrenaline (NA), and serotonin (5-HT) (ng/mg of wet tissue) in the amygdala in two-month-old rat offspring exposed to methylbenactyzine or ganglerone at different periods of prenatal development. \*p < 0.05 compared with the control group. For further details see caption to Fig. 1.

Changes of the neurotrasmitter status in the amygdala can affect functioning of other brain structures participating in regulation of sexual function, by means of neuronal connection of this structure with hippocampus and hypothalamus. It is shown that destruction of hypothalamo-amigdaloid connection, damage or irritation of the amygdala lead to neurohumoral alterations and sexual behavior abnormalities (Akmaev, 1993; Swanson, 1998; Dominguez, 2001). It is not excluded that optimization of integrative connections within limbic system and hypothalamus is extremely important concerning the effects of cholinergic system on sexual function.

Results of these researches show that pregnant females in "critical periods" of the embryo prenatal development exposure to M- and N-cholinoblockers can cause long-term changes in neurotransmitter systems activity in investigated structures of the rats brain in their postnatal life.

Exposure of M- and N-cholinolytics in the prenatal period leads to significant decrease of dopaminergic activity in the hippocampus and hypothalamus of rats offspring in comparison with control group. Results of various researches prove that defects of DA synaptic activity in the hippocampus at offspring can accompany to hippocampusassociated behavioral deficiencies at puberal individuals (Yanai, 1984; Smith, et al. 1986; Steingart et al., 2000).

Development of Male Sexual Function After Prenatal Modulation of Cholinergic System 109

studies, which have demonstrated that administration of an N-cholinolytic to pregnant females at different periods of gestation leads to long-term changes in the development of

The most significant changes in the concentrations of DA, 5-HT, and NA and their metabolites in brain structures were seen after administration of ganglerone at 9–11 and 12– 14 days of gestation, which was apparent as reductions in the synaptic activities of these brain neurotransmitter systems, particularly dopaminergic activity in the hippocampus and hypothalamus in two-month-old offspring of rats as compared with controls. Although 20 day embryos showed an increase in DA levels in response to administration of ganglerone at 12–14 days of gestation, this requires further investigation. It can be suggested that like nicotine, ganglerone, blocking N-cholinergic receptors, induced impairments to the formation and establishment of network systems in the developing embryonic brain, which promoted stable decreases in the synaptic activity of the transmitter systems of interest in the hippocampus and hypothalamus of twomonth-old offspring rats as compared with controls. This imbalance in neurotransmitter activity in brain limbic structures in twomonth-old offspring is a long-term neurochemical effect of prenatal treatment with

The neurotransmitter DA plays an important role in activating the sexual behavior in male rats; DA depletion in brain structures involved in regulating the behavioral states of body facilitates reductions in sexual activity (Gladkova, 2000; Mas et al., 1987; Pfaus & Phillips, 1991). Lesioning of different dopaminergic projections induces different behavioral syndromes depending on which part of the CNS is lesioned (Carey & Schwarting, 1986; Simon et al., 1986); in particular, damage to the dopaminergic projections of limbic

The noradrenergic and serotoninergic systems of the brain are also involved in regulating hormone-dependent behavioral states, including sexual behavior (Naumenko et al., 1983; Lenahan et al., 1986; Smeets & Reiner, 1994). Affecting the secretion of gonadoliberin in the hypothalamic nuclei, NA and 5-HT act on α2,β2-adrenoreceptors and 5-HT1,2 serotonin receptors to take part in the central regulation of the endocrine system of the male body and,

Thus, prenatal modulation of N-cholinergic brain mechanisms with ganglerone can alter the activities of DA, NA, and 5-HT systems, which are directly involved in regulating motivation and components of coitus in adult offspring. We believe that this mechanism is the main cause of long-term behavioral impairments in pubescent offspring subjected to prenatal exposure to cholinolytics. The prenatal effects of cholinolytics on sexual function in offspring represent a paradox, which is that in relation to the cholinergic system, sexual activity in adult males is regulated mainly by M-cholinolytic mechanisms, while during the

Another mechanism mediating the long-term actions of cholinolytics on sexual function in offspring consists of the involvement of endocrine factors. Considering the role of the central and peripheral compartments of the nervous system in controlling the hypothamalohypophyseal-gonadal system in males, it can be suggested that prenatal administration of cholinolytics to pregnant females might also have long-term consequences in relation to the

The results obtained from endocrine studies supported the occurrence of endocrine impairments in pubescent offspring (Fig. 8). A significant reduction in testosterone levels was seen in offspring subjected to prenatal exposure to ganglerone at different periods of

prenatal period, these are more dependent on the activity of N-cholinergic system.

brain neurotransmitter systems in 20-day embryos and two-month offspring of rats.

ganglerone, which in turn may facilitate sexual dysfunction in fertile males.

structures leads to impairments in male sexual function (Hull et al., 1984).

thus, in controlling male sexual function.

endocrine system of offspring.

The prenatal nicotine exposure which is exogenous ligand of N-cholinergic receptor leads to nonperishable change of activity of the basic neurotransmitter systems in brain structures in a postnatal period. The decrease of DA concentration in the brain structures at males has been detected at offspring which were subjected to prenatal exposure of N-cholinolytic ganglerone, and at females - to mainly prenatal exposure of M-cholinolytic methylbenactyzine (Genedani et al., 1983; Ribary, 1985; Lichtensteiger et al., 1988; Lichtensteiger & Schlumpf, 1993; Muneoka et al., 1997).

In spite of various mechanisms of M- and N-cholinolytics action in the organism, the longterm effects of these drugs on the basic transmitter systems development in offspring are basically the same, namely inhibiting a metabolism of these neurotransmitters. Prenatal exposure of selective M- and N-cholinolytics, like other chemical drugs and ecological toxicants with cholinergic properties, causes the long-term changes in programming of neurotransmitter functions in 2-month's offspring which, in turn, can participate in the development of neuro-behavioral anomalies, appetent and affective disturbances at puberal individuals (Lauder, 1985; Turlejski, 1996; Levitt et al., 1997; Dreyfus, 1998; Azmitia, 2001).

Thus, the prenatal exposure of M- and N-cholinolytics to pregnant females produces longterm neurochemical changes in the development of brain neuromediatory systems. In the studied brain structures, both males and females, exposure to prenatal cholinolytics, significant decrease of DA, 5-HT, NA concentrations and change of level of their metabolites were marked. Prenatal exposure to ganglerone N-cholinolytics to pregnant females on 9-19 gestational days exerts most appreciable long-term effect on neurotransmitter development, which leads to reduction of dopaminergic, noradrenergic and serotonergic activity in the hippocampus and hypothalamus of 2-month-old rats progenies in comparison with control group. Is was noted that prenatal exposure of cholinolytics on DA concentration in the brain structures at 2-month-old rats progenies causes sexual dimorphism. These results indicate that exposure to M- and N-cholinolytics during the critical periods of prenatal development (9-11 and 12-14 gestational days) results in long-term changes in development of neuromediatory systems in the brain structure, which participate in regulation of behavioral and neuroendocrinal functions of rats offsprings.

#### **6. Endocrinological consequences of prenatal exposure to N-cholinolytics in the rats offspring's**

The involvement of N-cholinergic mechanisms in behavioral impairments in male offspring is associated with the properties of N-cholinoreceptors, which are involved in the regulation of the catecholaminergic system of the CNS. N-cholinergic neurons are connected to different types of neurons in the brain, and activation of N-cholinoreceptors by endogenous (acetylcholine) or exogenous (nicotine) ligands modulates the release of the transmitters DA, NA, and 5-HT, depending on the type of cell (Retana, 1993; McGehee et al., 1995). Nicotinic receptors are abundant in the subcortical areas of the brain during early fetal development (Lisk & Greemvald, 1983), so embryonic exposure to nicotine damages noradrenergic and dopaminergic synaptic transmission in the brain (Navarro et al., 1988; Seidler et al., 1992).

It follows from this that the mechanism of prenatal exposure to the N-cholinolytic ganglerone on sexual function in offspring may be mediated by modulation of brain transmitter systems, including DA, NA, and 5-HT systems, which indirectly regulate the processes of motivation and components of coitus (Bitran & Hull, 1987; Gladkova, 2000; Mas et al., 1987; Pfaus & Phillips, 1991). This thesis is supported by results from neurochemical

The prenatal nicotine exposure which is exogenous ligand of N-cholinergic receptor leads to nonperishable change of activity of the basic neurotransmitter systems in brain structures in a postnatal period. The decrease of DA concentration in the brain structures at males has been detected at offspring which were subjected to prenatal exposure of N-cholinolytic ganglerone, and at females - to mainly prenatal exposure of M-cholinolytic methylbenactyzine (Genedani et al., 1983; Ribary, 1985; Lichtensteiger et al., 1988;

In spite of various mechanisms of M- and N-cholinolytics action in the organism, the longterm effects of these drugs on the basic transmitter systems development in offspring are basically the same, namely inhibiting a metabolism of these neurotransmitters. Prenatal exposure of selective M- and N-cholinolytics, like other chemical drugs and ecological toxicants with cholinergic properties, causes the long-term changes in programming of neurotransmitter functions in 2-month's offspring which, in turn, can participate in the development of neuro-behavioral anomalies, appetent and affective disturbances at puberal individuals (Lauder, 1985; Turlejski, 1996; Levitt et al., 1997; Dreyfus, 1998; Azmitia, 2001). Thus, the prenatal exposure of M- and N-cholinolytics to pregnant females produces longterm neurochemical changes in the development of brain neuromediatory systems. In the studied brain structures, both males and females, exposure to prenatal cholinolytics, significant decrease of DA, 5-HT, NA concentrations and change of level of their metabolites were marked. Prenatal exposure to ganglerone N-cholinolytics to pregnant females on 9-19 gestational days exerts most appreciable long-term effect on neurotransmitter development, which leads to reduction of dopaminergic, noradrenergic and serotonergic activity in the hippocampus and hypothalamus of 2-month-old rats progenies in comparison with control group. Is was noted that prenatal exposure of cholinolytics on DA concentration in the brain structures at 2-month-old rats progenies causes sexual dimorphism. These results indicate that exposure to M- and N-cholinolytics during the critical periods of prenatal development (9-11 and 12-14 gestational days) results in long-term changes in development of neuromediatory systems in the brain structure, which participate in regulation of behavioral

**6. Endocrinological consequences of prenatal exposure to N-cholinolytics in**

The involvement of N-cholinergic mechanisms in behavioral impairments in male offspring is associated with the properties of N-cholinoreceptors, which are involved in the regulation of the catecholaminergic system of the CNS. N-cholinergic neurons are connected to different types of neurons in the brain, and activation of N-cholinoreceptors by endogenous (acetylcholine) or exogenous (nicotine) ligands modulates the release of the transmitters DA, NA, and 5-HT, depending on the type of cell (Retana, 1993; McGehee et al., 1995). Nicotinic receptors are abundant in the subcortical areas of the brain during early fetal development (Lisk & Greemvald, 1983), so embryonic exposure to nicotine damages noradrenergic and dopaminergic synaptic transmission in the brain (Navarro et al., 1988; Seidler et al., 1992). It follows from this that the mechanism of prenatal exposure to the N-cholinolytic ganglerone on sexual function in offspring may be mediated by modulation of brain transmitter systems, including DA, NA, and 5-HT systems, which indirectly regulate the processes of motivation and components of coitus (Bitran & Hull, 1987; Gladkova, 2000; Mas et al., 1987; Pfaus & Phillips, 1991). This thesis is supported by results from neurochemical

Lichtensteiger & Schlumpf, 1993; Muneoka et al., 1997).

and neuroendocrinal functions of rats offsprings.

**the rats offspring's**

studies, which have demonstrated that administration of an N-cholinolytic to pregnant females at different periods of gestation leads to long-term changes in the development of brain neurotransmitter systems in 20-day embryos and two-month offspring of rats.

The most significant changes in the concentrations of DA, 5-HT, and NA and their metabolites in brain structures were seen after administration of ganglerone at 9–11 and 12– 14 days of gestation, which was apparent as reductions in the synaptic activities of these brain neurotransmitter systems, particularly dopaminergic activity in the hippocampus and hypothalamus in two-month-old offspring of rats as compared with controls. Although 20 day embryos showed an increase in DA levels in response to administration of ganglerone at 12–14 days of gestation, this requires further investigation. It can be suggested that like nicotine, ganglerone, blocking N-cholinergic receptors, induced impairments to the formation and establishment of network systems in the developing embryonic brain, which promoted stable decreases in the synaptic activity of the transmitter systems of interest in the hippocampus and hypothalamus of twomonth-old offspring rats as compared with controls. This imbalance in neurotransmitter activity in brain limbic structures in twomonth-old offspring is a long-term neurochemical effect of prenatal treatment with ganglerone, which in turn may facilitate sexual dysfunction in fertile males.

The neurotransmitter DA plays an important role in activating the sexual behavior in male rats; DA depletion in brain structures involved in regulating the behavioral states of body facilitates reductions in sexual activity (Gladkova, 2000; Mas et al., 1987; Pfaus & Phillips, 1991). Lesioning of different dopaminergic projections induces different behavioral syndromes depending on which part of the CNS is lesioned (Carey & Schwarting, 1986; Simon et al., 1986); in particular, damage to the dopaminergic projections of limbic structures leads to impairments in male sexual function (Hull et al., 1984).

The noradrenergic and serotoninergic systems of the brain are also involved in regulating hormone-dependent behavioral states, including sexual behavior (Naumenko et al., 1983; Lenahan et al., 1986; Smeets & Reiner, 1994). Affecting the secretion of gonadoliberin in the hypothalamic nuclei, NA and 5-HT act on α2,β2-adrenoreceptors and 5-HT1,2 serotonin receptors to take part in the central regulation of the endocrine system of the male body and, thus, in controlling male sexual function.

Thus, prenatal modulation of N-cholinergic brain mechanisms with ganglerone can alter the activities of DA, NA, and 5-HT systems, which are directly involved in regulating motivation and components of coitus in adult offspring. We believe that this mechanism is the main cause of long-term behavioral impairments in pubescent offspring subjected to prenatal exposure to cholinolytics. The prenatal effects of cholinolytics on sexual function in offspring represent a paradox, which is that in relation to the cholinergic system, sexual activity in adult males is regulated mainly by M-cholinolytic mechanisms, while during the prenatal period, these are more dependent on the activity of N-cholinergic system.

Another mechanism mediating the long-term actions of cholinolytics on sexual function in offspring consists of the involvement of endocrine factors. Considering the role of the central and peripheral compartments of the nervous system in controlling the hypothamalohypophyseal-gonadal system in males, it can be suggested that prenatal administration of cholinolytics to pregnant females might also have long-term consequences in relation to the endocrine system of offspring.

The results obtained from endocrine studies supported the occurrence of endocrine impairments in pubescent offspring (Fig. 8). A significant reduction in testosterone levels was seen in offspring subjected to prenatal exposure to ganglerone at different periods of

Development of Male Sexual Function After Prenatal Modulation of Cholinergic System 111

Experimental researches have shown that the rats males subjected to prenatal exposure of a ganglerone and characterized by low sexual activity, have appeared sensitive to effects of

**Intromissions**

**(n)**

**Control** 17,1 ±3,1 26,0 ±9,0 11,4 ±2,1 53,9 ±16,0 0,90 ±0,17 278,8 ±53,0 403,5 ±29,1

**latency of**

**1 hour** 29,6 ±1,3\* 13,3 ±2,0\* 17,1 ±2,9\* 29,5 ±3,2\* 1,83 ±0,22\* 184,4 ±36,4\* 313,4 ±41,2\*

**7 days** 14,1 ±2,1 21,9 ± 3,8 10,1 ±2,9 42,3 ±7,8 1,04 ±0,18 267,0 ±41,5 394,1 ±54,3

**1 hour** 24,6 ±2,8\* 12,4 ±2,5\* 18,8 ±3,1\* 28,7 ±4,7\* 1,55 ±0,19\* 198,3 ±36,0\* 329,1 ±39,5\*

**7 days** 16,6 ±2,1 30,5 ± 4,1 12,0 ± 2,4 55,8 ±7,2 1,10 ±0,17 288,2 ±41,5 386,0 ±50,6

**1 hour** 31,6 ±4,4\* 8,30 ±2,3\* 21,2 ±3,8\* 23,3 ±5,9\* 1,97 ±0,30\* 166,2 ±32,1\* 320,5 ±52,6\*

**7 days** 19,9 ±3,0 24,2 ± 3,6 12,2 ±1,2 45,4 ±6,7 1,09 ±0,18 237,3 ±38,2 425,0 ±57,9

Note: The sexual activity is recorded in 1 hour after an injection and for 7 days of an afteraction of

exposure to ganglerone on 12-14 day of gestation (G13 group) before and after application of

Cholinomimetic drug arecoline (2 mg/kg), galantamine with a ganglerone (accordingly, 1mg/kg and 5 mg/kg) and agonist of D1,D2-dopaminergic receptors apomorphinum (1 mg/kg) considerably enhanced sexual function. Components of sexual function - mounts, intromissions and ejaculations after pharmacological correction were high enough though did not reach in certain cases indexes of sexual behavior of control offspring. On the contrary, time components of a sexual behavior specified about sufficient high degree of

In spite of significant enchancement of the sexual function, the obtained data have shown that correction of sexual activity descended only during the period of drugs action - within 1 days. For 7 day after introduction of stimulating drugs quantitative and qualitative

Table 4.

**Mounts**

**(n)**

**latency of mount**

**(sec)**

**G13 group**

**Arecoline**

**Galantamine + Ganglerone**

**Apomorphinum**

the agents. (M±m).

drugs.

\*-p < 0.05 compared with control group.

sexual activation, including motivation enchancement.

characteristics of rats male sexual behavior were reverted on initial level.

gestation, with lowest values seen in the offspring of group G13 (2.4-fold decrease). There was also a significant decrease in the testosterone level in offspring subjected to prenatal exposure to methylbenactyzine in group M18. The LH and FSH levels in the blood of all groups were increased.

The hormonal-motivational component of sexual behavior of male rats is known to controlled at the central level by testosterone, which is metabolized to estradiol, while ejaculation is controlled at the peripheral level by the non-aromatized dihydrotestosterone and only partially by testosterone (Lisk, 1983). Low testosterone levels in G10–G13 offspring could therefore facilitate alterations in both the central motivational and the peripheral ejaculatory components of sexual behavior. The reduced testosterone level in G10–G18 offspring correlated with low sexual activity and, conversely, numbers of the sexually more active males of groups M10–M18 had higher testosterone levels.

Fig. 8. Serum Testosterone, LH and FSH levels in two-month-old rat offspring exposed to ganglerone or methylbenactyzine at different periods of prenatal development. \*p < 0.05 compared with control group. For further details see caption to Fig. 1.

Thus, along with neuronal factors, changes in the hormonal background probably represent a further cause of impairments to sexual functions in offspring subjected to prenatal exposure to central cholinolytics. Reproductive impairments induced by damage to the neuroendocrine and neurotransmitter systems during the fetal period of ontogenesis due to prenatal exposure to cholinolytics may in later life become the cause of impairments to the ability of males to mate and produce offspring.


**5. Performance characteristics**

In order to produce a reliable assessment of method performance, all necessary performance characteristics should be evaluated in carefully designed experiments. ICH guideline Q2R1 specifies which performance characteristics should be evaluated for validation. However, interpretation of the table for protein products is not straightforward. This is due in part to the fact that ICH Q2R1, Q6B and the industry used different nomenclature to describe the type of methods. The table below details the performance characteristics that should be assessed during qualification, and subsequently during the confirmatory validation experiments for protein products.

Analytical Method Validation for Biopharmaceuticals 123

Nearest Reporting Interval

due to the inability of bioassays to provide precision that would justify such a reporting interval. Therefore, bioassays with a standard deviation of 11.8 would result in a reporting interval of 1. Similarly, an HPLC assay with a standard deviation of 1.3 for the main peak would result in a reporting interval of 0.1. Reporting intervals for impurities (minor peaks) need to be consistent with reporting intervals for the main peak. In general, STD of equal or less than 2 (in units reported by the method) is required to ensure a reporting interval of one decimal place. The argument can be raised that for low level, minor analytes (for example, the dimer in SEC present at 1%), the requirement for STD to be at or below 2% is too generous. This will result in an RSD of 200% for the peak. In such an instance, this would indicate that the minor peak is well below the detection level, because theoretically the RSD at the LOD

level should not exceed 33% (Long and Winefordner 1983; Hayashi and Matsuda 1995)

obtained during qualification for protein products.

Horwitz and Albert 1997; Horwitz and Albert 1997)

where, C is the concentration of the analyte in mg/g.

fully suitable for the intended application.

concentration method), will be 16 and 2.8 %, respectively.

Standard Deviation (in reported units)

**Table 2.** Recommended Nearest Reporting Results Based on Standard Deviation

The table below proposes the nearest reporting intervals based on standard deviations

≤2.0 0.1 ≤20 1

Method precision is closely linked to the concentration of the analyte. The best-known relationship between analyte concentration and RSD is the Horwitz equation (Horwitz 1982;

1-0.5 ) logC( RSD = 2

Based on the Horwitz equation, the precision of the measurement, expressed as RSD,

The Horwitz relationship can provide good guidance for method precision targets during method development and qualification. Intermediate precision obtained during these studies should meet the variability derived from the Horwitz equation for each individual analyte. If, during execution of the qualification experiments, the precision of the measurements exceeds values derived from the Horwitz equation, this may indicate that the assay may need to be redeveloped, or that the technology utilized in the assay may not be

Typically, proteins are available for analysis as solutions, with concentrations ranging widely from 1 g/ml (e.g., a growth factor) to 100 mg/ml or higher (e.g., a monoclonal antibody). In such cases, expectations for the RSD of measurements of the main protein analyte in these solutions, based on the Horwitz relationship (e.g., using protein

doubles for each decrease of analyte concentration of two orders of magnitude.

\*In some cases may be required by USP <1225>.

**Table 1.** Performance Characteristics that need to be Evaluated During Qualification/Validation by Method Type


**5. Conclusions**

This study was conducted to explore asymmetries in local strength, vertical jumping and ROM, and to investigate whether these asymmetries are related to COD performance in healthy elite-basketball players. A substantial variability among asymmetries in di fferent tests was noted. This implies that coaches and physiotherapists should not rely exclusively on the <10 % threshold when they are deciding on the athletes return to play, or planning interventions for reducing asymmetries. In particular, it is expected for RTD asymmetries to be larger than maximal strength (i.e., peak torque) asymmetries. In the attempt to elucidate which asymmetries are more relevant for performance, specifically to the COD ability, we performed linear regressions which showed more than one type of asymmetry is should be considered in the analyses to su fficiently explain the COD performance. Notably, the best model for predicting COD performance included both maximal strength and RTD asymmetry, both hip and trunk asymmetry, one vertical jump asymmetry, one ROM asymmetry, as well as asymmetry in functional leg length. Therefore, interventions should likely target multiple types of asymmetries when trying to improve COD performance. We encourage practitioners to use a wide testing battery to test di fferent aspects on local and global level of the body to obtain a clearer picture of athletes' asymmetries.

**Author Contributions:** Conceptualization, F.U. and N.Š.; Data curation, F.U.; Formal analysis, F.U.; Funding acquisition, N.Š.; Investigation, F.U.; Methodology, N.Š.; Project administration, N.Š.; Resources, N.Š.; Software, N.Š.; Supervision, N.Š.; Validation, F.U. and N.Š.; Writing—original draft, F.U. All authors have read and agreed to the published version of the manuscript.

**Funding:** The study was supported by the Slovenian Research Agency through the project TELASI-PREVENT [L5-1845] (Body asymmetries as a risk factor in musculoskeletal injury development: studying etiological mechanisms and designing corrective interventions for primary and tertiary preventive care).

**Acknowledgments:** The authors would like to thank the athletes and management from the basketball clubs participating in this study. Additionally, many thanks to the research assistants who helped at testing.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.


**1. Introduction**

Over the years, and especially since the Second World War, land transformation has become more and more intense, leading to the overexploitation of land and to the progressive recognition of its finite nature. More recently, the COVID-19 crisis has further warned us about the importance that a present and future sustainable built, as well as natural, environment, could have in facing unexpected emergencies more resiliently [1]. There is, therefore, an increasing need to find and adopt integrated solutions to make present and future development more sustainable [2]. Thus, it is essential for policy and decision makers to take careful decisions on urbanization and land use management, approaching the latter not only as a political and technocratic decision but as one that affects our society's well-being and quality of life [3].

This perspective is well acknowledged at the European level, with the European Union (EU) which, through time, has introduced a growing number of policies and actions aiming at promoting a more sustainable approach to development and urbanization [4]. In particular, the EU is trying to halt excessive land transformation with its objective to achieve zero net land take by 2050 [5] and, more recently, the European Green Deal has stressed the need to make Europe climate neutral by 2050 [6]. As a result, in the last few years, policy and decision makers at all territorial levels have started to dedicate increasing efforts to pursue urbanization and land use models that are more sustainable, thus leading to the consolidation of an increasingly heterogeneous set of interventions and practices aiming at this direction [7]. At the same time, it should be noted that this has happened from both the top-down and the bottom-up levels, in the context of both urbanized and depopulated remote rural areas [8,9].

Research and studies on sustainable urbanization and land-use have also increased through time, often stemming from different definitions and interpretations of "sustain-

**Citation:** Solly, A.; Berisha, E.; Cotella, G. Towards Sustainable Urbanization. Learning from What's Out There. *Land* **2021**, *10*, 356. https://doi.org/10.3390/ land10040356

Academic Editor: Jefferey M. Sellers

Received: 22 February 2021 Accepted: 23 March 2021 Published: 1 April 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

ability". When focusing on the use of land, the most recent definitions of sustainable urbanization perceive the latter as the "design of future urban development as well as the re-development of existing ones in an environmentally friendly and resource-efficient manner" [10] (p. 1). In particular, sustainable land use seems to depend both on the socio-economic processes that trigger spatial development and on the effectiveness of the instruments that regulate these processes [3]. Adopting a similar perspective, the recently concluded ESPON SUPER project (Sustainable Urbanization and land-use Practices in European Regions, 2018–2020; https://www.espon.eu/super (accessed on 15 March 2021)) reviewed the multiple approaches put in place in different European cities and regions towards the achievement of a more sustainable urbanization, bearing in mind that there are no "right instruments" or "right targets" that could possibly fit all territorial contexts, also due to the high heterogeneity that characterizes the European continent in terms of socioeconomic development, administrative culture and spatial governance and planning [11,12].

The present contribution builds on the results of the SUPER project to develop guidance for decision and policy-makers aiming at promoting a more sustainable urbanization of their territories. It does this through a critical analysis of the rich set of practices collected throughout the project and, in particular, exploring the variable degree of success that characterizes interventions aiming at different goals, as well as adopting different types of instruments. After this introduction, the second section introduces the theoretical framework of the SUPER project, before section three describes the methodology that it adopted to collect and analyze sustainable urbanization and land use practices throughout Europe. Section four constitutes the core of the paper; it provides a quantitative overview of the collected interventions, in particular in relation to their localization and degree of success, to then discuss more in depth their results in relation to their scope and objectives (i.e., densification, regeneration, containment, governance and sectoral policies), as well as to the types of instruments that they have employed (i.e., visions and strategies, rules and legal devices, land use regulations, programs and projects). Finally, a concluding section rounds off the contribution, summarizing its main messages and the implications for decision and policy makers and introducing a number of avenues for future research.