Morphology of the testes of animals during estrogen injection in the prenatal period

. The aim of the study was to investigate the morphological changes in the testes of the offspring of white non-pedigreed laboratory mice when the synthetic oestrogen analogue synestrol was prenatally injected to the mother. After fertilization, the females were divided into 2 groups which received a single intramuscular injection of the synthetic oestrogen analogue synestrol on day E 11.5 of gestation at the same time of the day. The intact group was not exposed to any treatment. The experimental group was exposed to synestrol, a synthetic oestrogen analogue, as a 2% oil solution at a dose of 40 µg/kg (C-40). The results of morphometric analysis of testes of descendants of white outbred laboratory mice after prenatal single injection of synthetic oestrogen analogue synestrol in a dose of 40 µg/kg showed morphological changes in the organ parenchyma, manifested as decrease in mean number of Sertoli cells (C-40) 17.4±1.1 compared to intact group 20.8±1.9; decrease in mean number of spermatogonia (C-40) 24.4±1.1 compared to intact group, decrease in mean number of spermatozoa (C-40) 178.0±4.2 compared to intact group 196.6±5.3. There was observed a change in the endocrine apparatus of testes, expressed as a decrease in the mean area of Leydig cell nuclei in the intact group of 6,72±1,78. Prenatal effects of synestrol revealed in an experimental model, allow us to use it to search for means of postnatal developmental testicular dysfunction correction as well as to develop optimal doses of oestrogenic preparations during


Introduction
Scientists now have information about many environmental factors that cause reproductive dysfunction, but the true causes of male reproductive failure have not been conclusively established.
Environmental factors that have a negative impact on human and animal reproductive function are, for the most part, anthropogenic.These include chemical compounds used in production, agriculture and households and pharmaceuticals [1,2].
Experimental and clinical studies demonstrate the adverse effects of various perinatal risk factors on the formation and function of the reproductive system of the foetus and the neonate [3].
It is evident that the foundations for adult pathology are laid early in ontogenesis [4].Clinical studies of oestrogen use during pregnancy show delayed testicular formation, increased Leydig cell ratios, hypospadias and hypoplasia of the testes, cysts in the testicular appendage, atrophic changes in the seminal vesicles, prostate and testes [5].
In the field of science, the prenatal impact of hormones and their analogues on the future offspring has received particular attention.Increased hormone levels cause changes not only in the mother's body, but also in the forming foetus.Morphological studies in this direction will help to prevent pathological disorders of male reproductive function in postnatal ontogenesis.All this determines the creation of a biological model of estrogen influence in the embryonic period of progeny gonad formation.

Materials and methods
In this experimental study were used white non-pedigreed laboratory mice obtained from the "Laboratory Animal Nursery", located at: Republic of Bashkortostan, Chishminsky district, Gorny village.
Vivarium and animal housing conditions comply with RD-APK 3.10.07.02-09 "Guidelines for the housing of laboratory animals in the vivariums of research institutes and educational institutions", other sanitary norms and requirements of veterinary control and supervision of work with laboratory and experimental animals (license dated 25.01.2005 by the Federal Service for Surveillance in Healthcare and Social Development).
Female mice in oestrus were placed with sexually mature males for one day to initiate pregnancy.Pregnancy initiation was counted from 12 noon of the previous day to 12 noon of the following day.The first day of placement was considered day zero, the second day was considered the first day of gestation.
After fertility, the females were divided into 2 groups.The intact group (n = 15) was not exposed to any treatment.The experimental group (n = 15) received a single intramuscular injection of the synthetic oestrogen analogue synestrol as a 2% oil solution at a dose of 40 µg/kg (C-40) on gestational day E 11.5 at the same time of the day.All calculations and drug efficacy were performed according to the recommended ratios in the literature for converting doses of substances to µg/kg for mice [6][7][8].
All experimental manipulations have been carried out in accordance with the Geneva Convention «Internetional Guiding Principals for Biomedical Involving Animals» (Geneva, 1990) and the World Medical Association's Declaration of Helsinki Declaration on the Humane Treatment of Animals.
The offspring obtained after reaching reproductive age were bred from the experiment during the same phase, diestrus, on day 90 from the birth of the animals [9].Male mice were selected from all 2 groups of the obtained offspring.The testes of the offspring of white nonpedigreed laboratory mice were the object of the study.
Study of the material: For morphological studies, the obtained material was subjected to standard histological treatment.The fixed testes were cut uniformly in the centre of the organ and the structural components of the gonads were studied in a standard organ slice area [10].
Examination, visualisation and morphometry of histological preparations of testes of offspring were performed using an Axioobserver inverted biological microscope for laboratory research with a D1 tripod by Carl Zeiss Microscopy GmbH (Germany), with specialised software ZEN 2018 (the microscope was provided by the Cell Culture Laboratory of the Central Scientific Research Laboratory of Bashkir State Medical University.To count the structural tissue elements in the progeny gonads, a 90x immersion lens was used, at standard fields of view [10]. Histological preparations were photographed using an AxioCam MRc5 digital camera (ZEISS, Japan), at x100 magnification.Morphometric measurements of progeny testes structures were performed on the whole area of the organ slice, where the average parameters of the structures were measured.
Immunohistochemical analysis was performed on slices from paraffin blocks of testes of progeny intended for standard morphological examination.Paraffin sections were deparaffinised and rehydrated according to standard methods.Markers of proliferative activity of Ki-67 protein were determined by immunohistochemical method.The indirect streptavidin-biotin detection system Leica BOND (Novocastra™, Germany) for mouse (Mouse Monoclonal Antibody Ki-67 Antigen.Clone MIB-1; dilution: 1:300) was used according to the recommendation of the manufacturer Santa Cruz Biotechnology (USA).Histological sections 4 μm thick were stained using Leica Microsystems Bond™ immunohistostainer (Germany).The stained preparations were evaluated using a Leica light microscope, and the average number of immunopositive cells with a positive reaction was counted in 10 fields of view of each sample at a magnification of x100.The average number of cells positive to antigens was calculated by the ratio with the cells in which these antigens were not detected (per 100 counted cells).
Antibody expression was evaluated in the following cells of the testes of the offspring: spermatogonia, spermatocytes, spermatids, spermatozoa, and Leydig cells.
The research and care of the experimental animals was carried out in accordance with Directive 2010/63/EC of the European Parliament and Council 22/09/2010 on the protection of animals for scientific purposes and the recommendations of other international Russian and institutional rules in the field of bioethics.
Statistical processing was performed using Statistica 7.0 software (StatSoft, USA).The arithmetic mean and its standard error (M ± SD) were calculated for each parameter.Significance of the changes was assessed using Student's T-test, differences were determined at the achieved significance level P≤0.05.
There was a change in the endocrine apparatus of testes, expressed as a decrease in the mean area of Leydig cell nuclei in the intact group was 6.72±1.78(p≤0.05).
There was a slight decrease in the mean number of convoluted seminal tubules in the experimental group (C-40) 26.4±1.1 as compared to the intact group 28.8±2.6.The mean cross-sectional area of convoluted seminal tubule showed no change in the intact group was 1696.20±562.39, in the experimental group (C-40) 1651.23±442.The mean diameter of the The difference between the means with different letters in the same row is significant (P<0.05).The analysis of Ki-67 marker expression (Table 2) in the experimental group of C-40 positively stained cells in spermatogonia, compared to the intact group, showed no significant differences.Comparative analysis of the intact group with the experimental group of C-40 spermatocytes cells in the epithelium of the convoluted seminal tubule showed no differences.The expression level of the marker Ki-67 in spermatid cells in the epithelium of the convoluted seminal tubule in the experimental group C-40 increased by and 4.1%, respectively, compared to the intact group.No significant differences were found in positively stained spermatid cells in the lumen of the convoluted seminal tubule.The degree of Ki-67 protein expression decreased in the C-40 group in Leydig cells in the connective tissue layers between the convoluted seminal tubules by 48.0% (p ≤0.05).Leydig cells in the connective tissue stroma between the convoluted seminal tubules 5,0±1,0 2,4±0,5* *The difference between the means with different letters in the same row is significant (P<0.05).

Discussion
The obtained results of the prenatal effects of the synthetic oestrogen analogue synestrol at a dose of C-40 µg/kg (C-40) deepen and extend the knowledge about its damaging and negative effects on the morphofunctional state of the testes of offspring of white nonbred laboratory mice, which are manifested in disturbance of the generative apparatus as a decrease in the average number of Sertoli cells, a decrease in the average number of spermatogonia, a decrease in the average number of spermatozoa as compared with the intact group.Changes were also observed in the endocrine apparatus of the testes of the offspring, manifested by a decrease in the average area of Leydig cell nuclei.At present, quite a large amount of data has been accumulated on the negative prenatal effects of estrogens on mammalian reproductive function and mechanisms of genomic and non-genomic effects of estrogens on gonad development have been studied [11,12].

Conclusion
In conclusion, the prenatal effects of the synthetic estrogen analogue synestrol identified in the experimental model provide an opportunity to use it to search for means to correct postnatal developmental testicular dysfunction and to develop optimal doses of estrogen drugs administered during pregnancy.

Fig. 3 .Fig. 4 .
Fig. 3. Testis of the offspring of the offspring of the intact group.IHC reaction for Ki-67 marker.Dyeing of nuclei with haematoxylin.x100.

Table 1 .
convoluted seminal tubules in the intact group was 22.38±4.36and in the experimental group (C-40) 21.56±4.42.The mean thickness of spermatogenic epithelium in the intact group was 4.41±0.86, in the experimental group (C-40) 4.71±0.53.No significant changes were observed in the mean spermatocyte and spermatid count.Morphometric indices of the convoluted seminal tubules of the offspring of white mice without mice in prenatal single injection of the synthetic drug synestrol at a dose of C-40 µg/kg

Table 2 .
Number of Ki-67 immunopositive cells in testes of offspring of white mongrel laboratory mice during prenatal administration of synthetic preparation synestrol