animals

Article The Modulation of Functional Status of Bovine Spermatozoa by

Vitaly Denisenko 1, Irena Chistyakova 1 , Natalia Volkova 2 , Ludmila Volkova 2, Baylar Iolchiev 2 and Tatyana Kuzmina 1,*

1 Branch of Federal Research Center for Animal Husbandry Named after Academy Member L.K. Ernst, Russian Research Institute of Genetic and Breeding Farm Animals, 196601 Saint-Petersburg, Russia; [email protected] (V.D.); [email protected] (I.C.) 2 Federal Research Center for Animal Husbandry Named after Academy Member L.K. Ernst, 142132 Moscow, Russia; [email protected] (N.V.); [email protected] (L.V.); [email protected] (B.I.) * Correspondence: [email protected]; Tel.: +7-9213-92-19-47

Simple Summary: Progesterone is an endogenous steroid hormone, which can induce and/or reactions in semen of certain mammalian species. Our study aimed to investigate the effect of progesterone on the functional status of fresh bovine spermatozoa using a chlortetracy- cline fluorescent probe. Results showed that heparin induced capacitation in spermatozoa incubated with or without progesterone. The destruction of microfilaments by an inhibitor of cytochalasin D blocked the stimulating effect of heparin. Steroid hormone in mixture with prolactin stimulated the acrosome reaction in spermatozoa, which was blocked by an inhibitor of microtubule polymerization   (nocodazole). At the acrosome stage, prolactin provided the undergoing of acrosome reaction in male gametes. This effect was noted both in the presence and absence of progesterone and inhibited Citation: Denisenko, V.; Chistyakova, I.; Volkova, N.; Volkova, by nocodazole. The supplementation of dibutyryl cyclic adenosine monophosphate during the L.; Iolchiev, B.; Kuzmina, T. The acrosome reaction to progesterone-untreated spermatozoa did not cause changes in proportion of Modulation of Functional Status of acrosome-reacted cells. However, when progesterone was added during capacitation, a significant Bovine Spermatozoa by Progesterone. increase in the proportion of capacitated cells was noted, which was inhibited by nocodazole. Thus, Animals 2021, 11, 1788. https:// progesterone under the action of prolactin and dibutyryl cyclic adenosine monophosphate deter- doi.org/10.3390/ani11061788 mines the functional status of fresh spermatozoa, which indicates progesterone-modulating effect on the indicators of post-ejaculatory maturation of male gametes. Academic Editors: Alessandra Santillo and Maria Abstract: The aim of this study is to identify the effects of progesterone (PRG) on the capacitation Maddalena Di Fiore and the acrosome reaction in bovine spermatozoa. The fresh samples were incubated with and without capacitation inductors (heparin, dibutyryl cyclic adenosine monophosphate (dbcAMP)), Received: 22 April 2021 hormones (prolactin (PRL), PRG), inhibitors of microfilaments (cytochalasin D) and microtubules Accepted: 10 June 2021 (nocodazole) during capacitation and acrosome reactions. The functional status of spermatozoa was Published: 15 June 2021 examined using the chlortetracycline assay. Supplementation of heparin stimulated capacitation

Publisher’s Note: MDPI stays neutral in the presence and absence of PRG. Cytochalasin D blocked the stimulating effect of heparin on with regard to jurisdictional claims in capacitation. The addition of PRL during capacitation (without PRG) did not affect the functional published maps and institutional affil- status of spermatozoa, while in PRG-treated cells PRL stimulated the acrosome reaction. PRL (with iations. and without PRG) increased the acrosome reaction in capacitated cells. These PRL-dependent effects were inhibited by nocodazole. During the acrosome reaction, in presence of dbcAMP, PRG decreased the proportion of acrosome-reacted cells compared to PRG-untreated cells. This effect in PRG-treated cells was canceled in the presence of nocodazole. In conclusion, PRG under the action of PRL and

Copyright: © 2021 by the authors. dbcAMP determines the changes in the functional status of native sperm cells, which indicates PRG Licensee MDPI, Basel, Switzerland. modulating effect on the indicators of post-ejaculatory maturation of spermatozoa. This article is an open access article distributed under the terms and Keywords: progesterone; bull spermatozoa; capacitation; acrosome reaction conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Animals 2021, 11, 1788. https://doi.org/10.3390/ani11061788 https://www.mdpi.com/journal/animals Animals 2021, 11, 1788 2 of 11

1. Introduction Capacitation is a biochemical process, which includes changes in membrane proteins and lipids, ion fluxes, an increase in the level of cyclic adenosine monophosphate (cAMP) and protein phosphorylation [1]. Successful fertilization of the oocyte includes many biome- chanical and biochemical changes in spermatozoa when passing the oviductial tract and reaching the oocyte-cumulus complex [2]. Progesterone (PRG) is the main physiological activator of acrosome reaction, which is a Ca2+-dependent process [3]. Progesterone is secreted by cumulus cells and present in high concentration during ovulation in the follicu- lar fluid, where it can act on sperm cells before they bind to the [4]. The follicular fluid promotes the processes of capacitation and acrosome reaction in bull sperma- tozoa [5]. Progesterone is an endogenous steroid hormone, which can induce capacitation and/or acrosome reaction in semen of certain mammalian species, and sperm response to the progesterone is highly species-specific. It has been well established that progesterone induces the capacitation or acrosome reaction in horse and human spermatozoa [6,7], but the data on the effect of the progesterone on bovine sperm cells are contradictory. In some cases, the progesterone provides only the capacitation, or only the acrosome reaction in pre-capacitated spermatozoa, or induces both cellular processes [8–10]. It has been shown that the acrosome reaction occurs only in capacitated cells [8]. Progesterone activates the capacitation process; however, it has been noted that PRG-induced acrosome reaction also can happen [11]. Progesterone-induced changes are mediated by intracellular mechanisms associated with protein kinase C and voltage-gated Ca2+ channels [12]. By stimulating or inhibiting protein kinases A, C, G and tyrosine kinase, it has been shown that progesterone induces the acrosome reaction via tyrosine kinase and protein kinase C, but in horse sperm cells this process does not depend on protein kinases A, C and bicarbonate [13]. In contrast, in human sperm, the acrosome reaction is dependent on protein kinase A [14]. Calcium is also an important modulator of the capacitation and the acrosome reaction, probably participating as a key mediator in the exchange of information between the sperm and the egg [15]. Induction of the acrosome reaction by progesterone leads to an increase in calcium entry from the extracellular environment. The control of the function of voltage-gated Ca2+ channels, apparently, helps to prevent the premature acrosome reaction [16]. In bovine spermatozoa, heparin and dbcAMP are compounds that activate processes of capacita- tion [17,18], while PRL induce the acrosome reaction [19]. One of the main processes of capacitation is the dynamic transformation of the cytoskeleton, in particular, actin. Actin is the most widely known cytoskeleton protein, acting as a secondary messenger in signal transduction [20]. Actin polymerization occurs during capacitation in various mammalian species, including cattle [20,21]. Microtubules are also involved in intracellular processes that determine the acrosome reaction [19]. The aim of this study is to identify the effects of different hormones (progesterone, prolactin) on the postejaculate processes (capacitation and acrosome reaction) in bovine spermatozoa, and the role of microtubules and microfilaments in these modifications.

2. Materials and Methods 2.1. Chemicals All chemicals were obtained from Sigma-Aldrich Co. (Steinheim, Germany), unless otherwise indicated.

2.2. Location The present study was performed in the development biology laboratory of the All- Russian Research Institute of Genetics and Farm Animal Breeding of the Federal Research Center for Animal Husbandry, named after academy member L.K. Ernst (Saint Petersburg- Pushkin, 59◦4203500 N, 30◦270500 E). Animals 2021, 11, 1788 3 of 11

2.3. Semen Collection and Preparation One ejaculate was collected from each of the sixty Black-and-White bulls (aged 3–4 years old) just before the experiment. All sperm samples were derived from animals of breeding farm OJSC Nevskoe immediately after ejaculation. Fresh sperm was collected twice weekly, with no apparent changes in animal health or semen quality throughout the semen collection interval. Fresh ejaculates from three bulls were mixed and the obtained mixture was used for each experiment. In order to remove the seminal plasma, the obtained fresh sperm was twofold cen- trifuged at 300× g for 10 min in Sp-TALP medium consisting of 100 mM NaCl, 3.1 mM KCl, 25 mM NaHCO3, 0.3 mM NaH2PO4, 21.6 mM sodium lactate, 0.5 mM CaCl2, 0.4 mM MgCl2, 10 mM HEPES, 1 mM pyruvate and 0.1% polyvinyl alcohol (PVA, 30,000–70,000 Da).

2.4. Study Design 2.4.1. Induction of Capacitation After sperm dilution to a final concentration 50 × 106 sperm/mL, fresh samples were divided into sixteen equal aliquots. Eight aliquots were incubated in Sp-TALP medium with supplementation of 6 mg/mL bovine serum albumin (BSA), 0.5 mM CaCl2 and with and without different reagents-capacitation inductor (heparin), hormones (PRL, PRG) and inhibitors of actin microfilaments (cytochalasin D) and tubulin microtubules (nocodazole): 1—without any reagents (control), 2—with 5 µg/mL heparin (or 10 ng/mL PRL) [18,22]; 3—with 5 µg/mL heparin (or 10 ng/mL PRL) and 10 µM cytochalasin D [23]; 4—with 5 ‘µg/mL heparin (or 10 ng/mL PRL) and 10 µM nocodazole [24]; 5—with 1 mg/mL PRG (control) [7]; 6—with 1 mg/mL PRG, 5 µg/mL heparin (or 10 ng/mL PRL); 7—with 1 mg/mL PRG, 5 µg/mL heparin (or 10 ng/mL PRL) and 10 µM cytochalasin D; 8— with 1 mg/mL PRG, 5 µg/mL heparin (or 10 ng/mL PRL) and 10 µM nocodazole. The ◦ incubation was performed in the atmosphere of 5% CO2 at 38.5 C and 95% humidity for 4 h.

2.4.2. Induction of Acrosome Reaction Acrosome reaction in spermatozoa, preliminary capacitated with 5 µg/mL heparin, was performed using 100 µg/mL lysophosphatidylcholine [25] and incubated in Sp-TALP medium with supplementation of 6 mg/mL BSA, 0.5 mM CaCl2 and with and without different reagents-capacitation inductor (dibutyryl cyclic adenosine monophosphate, db- cAMP), hormones (PRL, PRG) and inhibitors of actin microfilaments (cytochalasin D) and tubulin microtubules (nocodazole): 1—without any reagents (control), 2—with 100 µM dbcAMP (or 10 ng/mL PRL) [17]; 3—with 100 µM dbcAMP (or 10 ng/mL PRL) and 10 µM cytochalasin D; 4–with 100 µM dbcAMP (or 10 ng/mL PRL) and 10 µM nocodazole; 5— with 1 mg/mL PRG (control); 6—with 1 mg/mL PRG, 100 µM dbcAMP (or 10 ng/mL PRL); 7—with 1 mg/mL PRG, 100 µM dbcAMP (or 10 ng/mL PRL) and 10 µM cytochalasin D; 8—with 1 mg/mL PRG, 100 µM dbcAMP (or 10 ng/mL PRL) and 10 µM nocodazole. The ◦ incubation was performed in the atmosphere of 5% CO2 at 38.5 C and 95% humidity for 30 min (Figure1). Animals 2021, 11, 1788 4 of 11 Animals 2021, 11, x 4 of 12

Figure 1. Study design. Figure 1. Study design. 2.5. CTC Fluorescence Assay 2.5. CTC Fluorescence Assay µ ChlortetracyclineChlortetracycline (CTC) (CTC) was was dissolved dissolved at at750 750 µM Min ina buffer a buffer of 20 of mM 20 mM Tris, Tris, 130 130mM mM NaClNaCl and 5 mM L L-cysteine,-cysteine, and and the the pH pH was was adjusted adjusted to to7.8. 7.8. The The solution solution was was kept kept in a in a ◦ µ light-shieldedlight-shielded contain containerer at at 4 4°CC.. At At the the time time of ofthe the assay, assay, 20 20µL ofL ofthe the sperm sperm suspension suspension ofof each sample sample was was mixed mixed with with 20 µL 20 µofL the of CTC the CTCsolution solution and incubated and incubated with CTC with at 38.5 CTC at ◦ 38.5°C forC 10 for min. 10 min.Then, Then, 10 µL 10of µ25%L of glutaraldehyde 25% glutaraldehyde in 1 mM in Tris 1 mM (pH Tris 7.4) (pH was 7.4) added was to added a tofinal a final concentration concentration of 0.1% of glutaraldehyde 0.1% glutaraldehyde in each insample each for sample fixation. for A fixation. drop of Asperm drop of spermsuspension suspension (10 µL) w (10as µplacedL) was on placed a glass onslide a glassand mixed slide with and mixed10 µL of with 0.22 10M 1,4µL-diazo- of 0.22 M 1,4-diazobicyclobicyclo [2.2.2] octane [2.2.2] dissolved octane dissolved in glycerol in/ glycerol/phosphatephosphate buffered saline buffered (9:1) saline at room (9:1) tem- at room temperature.perature. Then, Then, coverslip coverslip was attached, was attached, drops drops of colorless of colorless nail polish nail polishwere applied were applied to fix to ◦ fixits itsedges. edges. The The slides slides were were stored stored in a light in a light-shielded-shielded container container at 4 °C at before 4 C beforescoring. scoring. TheThe slides slides were were scored scored with with a ZEISS a ZEISS AxioLab. AxioLab. A1. (KarlA1. (Karl Zeiss, Zeiss, Jena, Germany)Jena, Germany) equipped withequipped phase with contrast phase and contrast epifluorescence and epifluorescence optics. The optics. excitation The excitation for CTC for was CTC 380–400 was 38 nm,0– the emission–530400 nm, the emission nm. The–530 samples nm. The were samples analyzed were inanalyzed accordance in accordance with one with of the one three of the types ofthree CTC types fluorescence of CTC fluorescence patterns [26 patterns]: the uniform [26]: the fluorescenceuniform fluorescence in the entire in the sperm entire headsperm with uncapacitatedhead with uncapacitated cells, the fluorescence-free cells, the fluorescence band- infree the band post-acrosome in the post-acrosome region; capacitated region; ca- cells, Animals 2021, 11, x thepac absenceitated cells, of fluorescence the absence of in fluorescence the entire sperm in the head, entire with sperm the exceptionhead, with of the a thinexception bright5 of 12 of band

ofa thin fluorescence bright band in the of fluorescence equatorial area; in the and equatorial acrosome-reacted area; and cellsacrosome (Figure-reacted2). cells (Fig- ure 2).

2+ FigureFigure 2. Fluorescence 2. Fluorescence intensity intensity of of CTC-CaCTC-Ca2+-membrane-membrane-bound-bound complex complex in bull in spermatozoa bull spermatozoa was detected was by detected CTC fluo- by CTC fluorescencerescence probe. probe. ( A(A)) non-capacitatednon-capacitated sperm sperm cell; cell; (B ()B capacitated) capacitated sperm sperm cell; cell;(C) acrosome (C) acrosome-reacted-reacted sperm spermcell. Scale cell. bar Scale: 7 bar: 7 µm.µm. The The fluorescence fluorescence patterns patterns of of a a minimum minimum ofof 200200 sperm were were scored scored in ineach each exper experimentalimental group. group. All slides All slides were werekept kept at room temperature in light-shielded containers until examination. The sample on the slide was divided into quadrants at room temperature in light-shielded containers until examination. The sample on the slide was divided into quadrants with 6–8 separate fields being examined per quadrant. with 6–8 separate fields being examined per quadrant. 2.6. Statistical Analysis Results of the present study are predominantly presented using descriptive statistics. Average percent of sperm cells (in capacitation stage and acrosome reaction stage) in con- trol and experimental groups were compared by Student’s t-test, with data presented as means ± SEM. Results were considered significant when p < 0.05; p < 0.01; p < 0.001. Statis- tical analysis of the results was carried out using Statistic 7.0 package (StatSoft, Tulsa, OK, USA, 2016).

3. Results 3.1. The Effect of PRG on Functional Status of Bull Spermatozoa during Capacitation The analysis of effects of 1 µg/mL PRG on the capacitation process of bull spermato- zoa stimulated by heparin (5 µg/mL) is presented in Figure 3. The use of the chlortetracy- cline probe allows for visualization of the redistribution of Ca2+ in the sperm membrane by forming CTC-Ca2+-membrane-bound fluorescence complexes. The sperm cells, which were incubated for 4 h with heparin showed an increase of capacitated cells (26%, p < 0.001) and a decrease of acrosome-reacted spermatozoa (69%, p < 0.001) compared to intact untreated cells incubated for 4 h (16% and 81%, respectively, p < 0.001). The treatment with an inhibitor of microfilaments (cytochalasin D) at the concentration of 10 µM resulted in a lower percent of capacitated cells and a high proportion of acrosome-reacted sperm cells (12% and 83%, respectively, p < 0.001) versus those of cells treated with heparin only. The treatment of sperm with 10 µM inhibitor of microtubules (nocodazole) did not affect the cell ratio with different fluorescence pattern (25% vs. 69%, respectively, p < 0.001) com- pared to the group incubated with heparin. It should be noted that the effects of a single use of heparin or a mixture with PRG on spermatozoa were similar–an increase in the number of capacitated cells and a decrease in acrosome-reacted spermatozoa (26% and 23% vs. 69% and 70%, respectively). The usage of the inhibitor cytochalasin D stimulated the decrease of acrosome-reacted cells in PRG-treated group of spermatozoa during ca- pacitation (83%, p < 0.01). Animals 2021, 11, 1788 5 of 11

2.6. Statistical Analysis Results of the present study are predominantly presented using descriptive statistics. Average percent of sperm cells (in capacitation stage and acrosome reaction stage) in control and experimental groups were compared by Student’s t-test, with data presented as means ± SEM. Results were considered significant when p < 0.05; p < 0.01; p < 0.001. Statistical analysis of the results was carried out using Statistic 7.0 package (StatSoft, Tulsa, OK, USA, 2016).

3. Results 3.1. The Effect of PRG on Functional Status of Bull Spermatozoa during Capacitation The analysis of effects of 1 µg/mL PRG on the capacitation process of bull spermatozoa stimulated by heparin (5 µg/mL) is presented in Figure3. The use of the chlortetracycline probe allows for visualization of the redistribution of Ca2+ in the sperm membrane by forming CTC-Ca2+-membrane-bound fluorescence complexes. The sperm cells, which were incubated for 4 h with heparin showed an increase of capacitated cells (26%, p < 0.001) and a decrease of acrosome-reacted spermatozoa (69%, p < 0.001) compared to intact untreated cells incubated for 4 h (16% and 81%, respectively, p < 0.001). The treatment with an inhibitor of microfilaments (cytochalasin D) at the concentration of 10 µM resulted in a lower percent of capacitated cells and a high proportion of acrosome-reacted sperm cells (12% and 83%, respectively, p < 0.001) versus those of cells treated with heparin only. The treatment of sperm with 10 µM inhibitor of microtubules (nocodazole) did not affect the cell ratio with different fluorescence pattern (25% vs. 69%, respectively, p < 0.001) compared to the group incubated with heparin. It should be noted that the effects of a single use of heparin or a mixture with PRG on spermatozoa were similar—an increase in the number of capacitated cells and a Animals 2021, 11, x decrease in acrosome-reacted spermatozoa (26% and 23% vs. 69% and 70%, respectively).6 of 12 The usage of the inhibitor cytochalasin D stimulated the decrease of acrosome-reacted cells in PRG-treated group of spermatozoa during capacitation (83%, p < 0.01).

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Figure 3. The effect of 1 µg/mL PRG on capacitation process of bull spermatozoa stimulated by heparin (5 µg/mL) Figure 3. The effect of 1 µg/mL PRG on capacitation process of bull spermatozoa stimulated by heparin (5 µg/mL) (total (total number of counted cells-6400; number of experiments-4). 1—without any reagents (control), 2—with 5 µg/mL number of counted cells-6400; number of experiments–4). 1–without any reagents (control), 2–with 5 µg/mL heparin; 3– heparin; 3—with 5 µg/mL heparin and 10 µM cytochalasin D; 4—with 5 µg/mL heparin and 10 µM nocodazole; 5— with with 5 µg/mL heparin and 10 µM cytochalasin D; 4–with 5 µg/mL heparin and 10 µM nocodazole; 5– with 1 mg/mL PRG 1(control); mg/mL 6 PRG–with (control); 1 mg/mL 6—with PRG, 5 1 µg mg/mL/mL heparin; PRG, 5 µ7–g/mLwith 1 heparin; mg/mL 7—withPRG, 5 µg 1 mg/mL/mL heparin PRG, and 5 µ g/mL10 µM heparin cytochalasin and 10 D;µ M8– cytochalasinwith 1 mg/mL D; PRG, 8—with 5 µg 1/mL mg/mL heparin PRG, and 5 µ 10g/mL µM nocodazole heparin and. The 10 µ differencesM nocodazole. are significant The differences (percent are significantratio of capacitated (percent ratioand acrosome of capacitated-reacted and cells) acrosome-reacted a:b; a:d; b:c; c:d; c:b; b:j; cells)d:j; a:f; c:f;a:b; f:j at a:d; p < b:c; 0.001; c:d; c:b; b:d; b:j; d:e; d:j; a:h; a:f; e:f; c:f;f:j; e:h; f:j j:hat atp p< < 0.001; 0.01 (Studentb:d; d:e; a:h;’s e:f;t-test). f:j; e:h; j:h at p < 0.01 (Student’s t-test). The effect ofof PRLPRL (10(10 ng/mL)ng/mL) on appearance of bovine spermatozoaspermatozoa with differentdifferent fluorescencefluorescence patterns is shown in Figure4 4.. NoNo significantsignificant changeschanges inin thethe ratio ratio of of sperm sperm cells with different functional status was observed during the incubation period of cells with 10 ng/mL PRL (without PRG) for 4 h in comparison with capacitation of spermatozoa without PRL. The treatment of cells with cytochalasin D in PRG-free medium promoted the growth in number of acrosome-reacted cells during the 4 h incubation of spermatozoa with PRL in comparison with the sperm cells incubated 4 h with any reagents (82% vs. 63%, respectively, p < 0.001). The treatment with PRG caused changes in the biological action of PRL on sperm cells during incubation period; as well, the stimulatory effect on the growth of acrosome-reacted sperm cells was blocked by the microtubule inhibitor, nocodazole (81% and 82% vs. 70%, respectively, p < 0.01).

Animals 2021, 11, 1788 6 of 11

cells with different functional status was observed during the incubation period of cells with 10 ng/mL PRL (without PRG) for 4 h in comparison with capacitation of spermatozoa without PRL. The treatment of cells with cytochalasin D in PRG-free medium promoted the growth in number of acrosome-reacted cells during the 4 h incubation of spermatozoa with PRL in comparison with the sperm cells incubated 4 h with any reagents (82% vs. 63%, respectively, p < 0.001). The treatment with PRG caused changes in the biological action of

Animals 2021, 11, x PRL on sperm cells during incubation period; as well, the stimulatory effect on the growth7 of 12 of acrosome-reacted sperm cells was blocked by the microtubule inhibitor, nocodazole (81% and 82% vs. 70%, respectively, p < 0.01).

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Figure 4. The effect of PRL (10 ng/mL) on appearance of bovine spermatozoa with different fluorescence patterns (total numberFigure 4. of The counted effect cells-6400;of PRL (10 numberng/mL) ofon experiments-4).appearance of bovine 1—without spermatozoa any reagents with different (control); fluoresce 2—withnce 10 patterns ng/mL PRL;(total 3—withnumber 10of ng/mLcounted PRL cells and-6400; 10 µnumberM cytochalasin of experiments D; 4—with–4). 1 10–without ng/mL any PRL reagents and 10 µ (control)M nocodazole;; 2–with 5—with 10 ng/mL 1 mg/mL PRL; 3– PRGwith 10 ng/mL PRL and 10 µM cytochalasin D; 4–with 10 ng/mL PRL and 10 µM nocodazole; 5–with 1 mg/mL PRG (control); (control); 6—with 1 mg/mL PRG, 10 ng/mL PRL; 7—with 1 mg/mL PRG, 10 ng/mL PRL and 10 µM cytochalasin D; 8— 6–with 1 mg/mL PRG, 10 ng/mL PRL; 7–with 1 mg/mL PRG, 10 ng/mL PRL and 10 µM cytochalasin D; 8–with 1 mg/mL with 1 mg/mL PRG, 10 ng/mL PRL and 10 µM nocodazole. The differences are significant (percent ratio of capacitated and PRG, 10 ng/mL PRL anda:c; 10 a:b; µM c:d; a:f;nocodazole a:j; b:c; b:f; b:j;. The d:c; d:f;differences d:j; h:f; h:j are significantd:e; a:e;(percent a:h; b:e; b:h;ratio d:h of capacitated and acrosome- acrosome-reactedreacted cells) a:c; a:b; cells) c:d; a:f; a:j; b:c; b:f; b:j; d:c; d:f; d:j; h:f; h:j at p < 0.001; d:e; a:e; a:h;at b:e;p < b:h; 0.001; d:h at p < 0.01 (Student’s tat-test).p < 0.01 (Student’s t-test).

3.2.3.2. TheThe EffectEffect ofof PRGPRG onon FunctionalFunctional StatusStatus ofof BullBull SpermatozoaSpermatozoa duringduring AcrosomeAcrosome ReactionReaction TheThe effecteffect ofof 1010 ng/mLng/mL PRL on the time course of acrosomeacrosome reactionreaction inin bovinebovine sper-sper- matozoamatozoa is shown shown in in Figure Figure 55. .The The incubation incubation of ofcells cells with with PRL, PRL, without without PRG, PRG, during dur- the ingacrosome the acrosome reaction reaction period resulted period resultedin a high in percent a high of percent acrosome of acrosome-reacted-reacted cells (81% cells, p < (81%,0.001)p in< comparison 0.001) in comparison with intact with and intact PRG- andtreated PRG-treated control groups control (70 groups% and (70%67%, p and < 0.001). 67%, pFurthermore,< 0.001). Furthermore, the PRL-induced the PRL-induced effect on stimulation effect on stimulation of the acrosome of the acrosomereaction process reaction in processspermatozoa in spermatozoa was inhibited was inhibitedby nocodazole by nocodazole (70%, p < (70%,0.001)p versus< 0.001) PRL versus-treated PRL-treated PRG-un- PRG-untreatedtreated group (81 group%, p (81%,< 0.001).p < The 0.001). incubation The incubation of sperm of cells sperm with cells PRL with or the PRL mixture or the mix-with turePRG, with stimulated PRG, stimulated an undergoing an undergoing of acrosome of acrosome reaction reaction in capacitated in capacitated spermatozoa spermatozoa (81% (81%and 82 and%, 82%,respectively) respectively) in contrast in contrast with both with control both control groups, groups, which which was canceled was canceled under under the action of nocodazole (70% and 69%, respectively, p < 0.001). the action of nocodazole (70% and 69%, respectively, p < 0.001). The effect of PRG on the acrosome reaction process activated by 100 µM dbcAMP presented in Figure6. The addition of dbcAMP to intact PRG-untreated sperm cells 120 had no effect on the time course of the acrosome reaction (78%, respectively, p < 0.001) a comparedb to intactc and PRG-treatedd controle groupsf (78% andj 78%, respectively).h The 100 pre-incubation of cells with PRG and subsequent exposure to dbcAMP increased the number of capacitated cells compared to the spermatozoa incubated with dbcAMP only 80 (29% vs. 19, respectively, p < 0.001). During incubation of PRG-treated spermatozoa with the cytoskeleton-depolymerized agent cytochalasin D, no significant changes in the ratio of 60 sperm cells with a different functional status caused by exposure to dbcAMP were observed status 40

20 % cells withdifferent functional 0 1 2 3 4 5 6 7 8 Non-capacitated Capacitated Acrosome-reactive

Figure 5. The effect of 10 ng/mL PRL on the time course of acrosome reaction in bovine spermatozoa (total number of counted cells-6400; number of experiments–4). 1–without any reagents (control); 2–with 10 ng/mL PRL; 3–with 10 ng/mL Animals 2021, 11, x 7 of 12

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Figure 4. The effect of PRL (10 ng/mL) on appearance of bovine spermatozoa with different fluorescence patterns (total number of counted cells-6400; number of experiments–4). 1–without any reagents (control); 2–with 10 ng/mL PRL; 3–with 10 ng/mL PRL and 10 µM cytochalasin D; 4–with 10 ng/mL PRL and 10 µM nocodazole; 5–with 1 mg/mL PRG (control); 6–with 1 mg/mL PRG, 10 ng/mL PRL; 7–with 1 mg/mL PRG, 10 ng/mL PRL and 10 µM cytochalasin D; 8–with 1 mg/mL PRG, 10 ng/mL PRL and 10 µM nocodazole. The differences are significant (percent ratio of capacitated and acrosome- reacted cells) a:c; a:b; c:d; a:f; a:j; b:c; b:f; b:j; d:c; d:f; d:j; h:f; h:j at p < 0.001; d:e; a:e; a:h; b:e; b:h; d:h at p < 0.01 (Student’s t-test).

3.2. The Effect of PRG on Functional Status of Bull Spermatozoa during Acrosome Reaction The effect of 10 ng/mL PRL on the time course of acrosome reaction in bovine sper- matozoa is shown in Figure 5. The incubation of cells with PRL, without PRG, during the acrosome reaction period resulted in a high percent of acrosome-reacted cells (81%, p < 0.001) in comparison with intact and PRG-treated control groups (70% and 67%, p < 0.001). Furthermore, the PRL-induced effect on stimulation of the acrosome reaction process in Animals 2021, 11, 1788 spermatozoa was inhibited by nocodazole (70%, p < 0.001) versus PRL-treated PRG7 of-un- 11 treated group (81%, p < 0.001). The incubation of sperm cells with PRL or the mixture with PRG, stimulated an undergoing of acrosome reaction in capacitated spermatozoa (81% (25%and and82% 68%, respectively) and 29% and in 64%,contrast respectively, with bothp

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PRL and 1060 µM cytochalasin D; 4–with 10 ng/mL PRL and 10 µM nocodazole; 5–with 1 mg/mL PRG (control); 6–with

mg/mL PRG,status 10 ng/mL PRL; 7–with 1 mg/mL PRG, 10 ng/mL PRL and 10 µM cytochalasin D; 8–with 1 mg/mL PRG, 10 ng/mL PRL40 and 10 µM nocodazole. The differences are significant (percent ratio of capacitated and acrosome-reacted cells) a:b; a:c; a:f; a:j; d:b; d:e; d:f; d:j; e:b; e:c; e:f; e:j; h:b; h:e; h:f; h:j at p < 0.001 (Student’s t-test). 20 The effect of PRG on the acrosome reaction process activated by 100 µM dbcAMP

% cells withdifferent functional presented in Figure 6. The addition of dbcAMP to intact PRG-untreated sperm cells had 0 1 no effect2 on the time3 course4 of the acrosome5 reaction6 (78%, respectively,7 8 p < 0.001) com- pared to intact and PRG-treated control groups (78% and 78%, respectively). The pre-in- cubationNon-capacitated of cells with PRGCapacitated and subsequentAcrosome-reactive exposure to dbcAMP increased the number of

Figure 5. The effect of 10 ng/mLcapacitated PRL on cells the time compared course of to acrosome the spermat reactionozoa in incubated bovine spermatozoa with dbcAMP (total only number (29% of vs. 19, Figure 5. The effect of 10 ng/mL PRL on the time course of acrosome reaction in bovine spermatozoa (total number of counted cells-6400; number ofrespectively, experiments-4). p 1—without< 0.001). During any reagents incubation (control); of PRG 2—with-treated 10 ng/mL spermatozoa PRL; 3—with with 10 the ng/mL cytoskel- counted cells-6400; number of experiments–4). 1–without any reagents (control); 2–with 10 ng/mL PRL; 3–with 10 ng/mL PRL and 10 µM cytochalasin D;eton 4—with-depolymeri 10 ng/mLzed PRL agent and cytochalasin 10 µM nocodazole; D, no 5—with significant 1 mg/mL changes PRG (control);in the ratio 6—with of sperm mg/mL PRG, 10 ng/mL PRL;cells 7—with with 1 a mg/mL different PRG, functional 10 ng/mL status PRL andcaused 10 µ byM cytochalasinexposure to D; dbcAM 8—withP were 1 mg/mL observed PRG, (25% 10 ng/mL PRL and 10 µM nocodazole.and 68% and The 29 differences% and 64 are%, significantrespectively, (percent p < 0.001), ratio of whereas capacitated the andnocodazole acrosome-reacted promoted the cells) a:b; a:c; a:f; a:j; d:b; d:e; d:f; d:j;increase e:b; e:c; e:f; e:j;of h:b;acrosome h:e; h:f; h:j-atreactedp < 0.001 sperm (Student’s cells (15t-test).% and 78%, respectively, p < 0.001).

120 a b c d e f j h 100

80

60 status 40

20

% of cells withdifferent functional 0 1 2 3 4 5 6 7 8 Non-capacitated Capacitated Acrosome-reactive

Figure 6. The effect of PRG on the acrosome reaction process activated by 100 µM dbcAMP (total number of counted Figure 6. The effect of PRG on the acrosome reaction process activated by 100 µM dbcAMP (total number of counted cells- cells-6400; number of experiments-4). 1—without any reagents (control); 2—with 100 µM dbcAMP; 3—with 100 µM 6400; number of experiments–4). 1–without any reagents (control); 2–with 100 µМ dbcAMP; 3–with 100 µМ dbcAMP and dbcAMP and 10 µM cytochalasin D; 4—with 100 µM dbcAMP and 10 µM nocodazole; 5—with 1 mg/mL PRG (control); 10 µM cytochalasin D; 4–with 100 µМ dbcAMP and 10 µM nocodazole; 5–with 1 mg/mL PRG (control); 6–with mg/mL µ µ µ 6—withPRG, 100 mg/mL µМ dbcAMP; PRG, 100 7–withM dbcAMP; 1 mg/mL 7—withPRG, 100 1 µМ mg/mL dbcAMP PRG, and 100 10 µMM dbcAMP cytochalasin and 10D; 8–Mwith cytochalasin 1 mg/mL PRG, D; 8—with 100 µМ 1dbcAMP mg/mL PRG,and 10 100 µMµM nocodazole. dbcAMP and The 10 differencesµM nocodazole. are significant The differences (percent are ratio significant of capacitated (percent and ratio acros ofome capacitated-reacted andcells) acrosome-reacteda:f; b:f; c:f; d:f: e:f; h:f at p cells) < 0.001;a:f; b:f;a:j; b:j; c:f; c:j; d:f; d:j; e:f;e:j; h:j h:f atat p p< <0.01 0.001; (Studenta:j; b:j; c:j;’s t d:j;-test). e:j; h:j at p < 0.01 (Student’s t-test).

4.4. Discussion Discussion InIn our our experiments,experiments, inin the absence of of PRG, PRG, heparin heparin and and dbcAMP dbcAMP stimulated stimulated the the ca- capacitationpacitation and and did did not not affect affect the theprocess process of acrosome of acrosome reaction reaction in fresh in freshsperm. sperm. PRL worked PRL workedin the opposite in the opposite way–it way—itincreased increased the proportion the proportion of acrosome of acrosome-reacted-reacted spermatozoa spermatozoa and did andnot didhave not an have effect an on effect cells on at cellsthe capacitation at the capacitation stage. stage. During capacitation, actin polymerization occurs in mammalian spermatozoa. Before the acrosome reaction, actin-cleaving proteins are activated, which leads to actin depoly- merization [27]. Actin polymerization is one of the mechanisms dependent on capacita- tion that protects sperm from premature acrosome reaction [28]. Polymerization of micro- tubules occurs during the acrosome reaction, which is necessary for the normal fertiliza- tion process [29]. It can be assumed that the action of compounds, which activate the pas- sage of capacitation, should be associated with the polymerization of actin; compounds that stimulate the polymerization of microtubules are suitable to stimulate the acrosome reaction. According to our results, the heparin-stimulated capacitation is associated with Animals 2021, 11, 1788 8 of 11

During capacitation, actin polymerization occurs in mammalian spermatozoa. Be- fore the acrosome reaction, actin-cleaving proteins are activated, which leads to actin depolymerization [27]. Actin polymerization is one of the mechanisms dependent on capacitation that protects sperm from premature acrosome reaction [28]. Polymerization of microtubules occurs during the acrosome reaction, which is necessary for the normal fertilization process [29]. It can be assumed that the action of compounds, which acti- vate the passage of capacitation, should be associated with the polymerization of actin; compounds that stimulate the polymerization of microtubules are suitable to stimulate the acrosome reaction. According to our results, the heparin-stimulated capacitation is associated with the functioning of actin cytoskeleton, since the depolymerization of micro- filaments mediated by cytochalasin D, caused the decrease in the proportion of capacitated sperm cells. In buffalo spermatozoa during capacitation, dbcAMP also stimulates actin polymerization [30]. Microtubules are necessary for implementation of effect of PRL during the acrosome reaction period, since the simultaneous use of the inhibitor nocodazole led to the dramatic drop in the proportion of acrosome-reacted cells in bovine spermatozoa. In breast cancer cells, prolactin promotes actin regulation, thus enhancing cell movement [31]. Also, microtubules can participate in the transmission of the prolactin signal from the receptor to the genes of milk protein [32]. The usage of homological follicular fluid for incubation of bovine spermatozoa in various concentrations, containing a large amount of PRG, promoted sperm capacitation and induced the acrosome reaction [5]. However, the data on the effect of PRG on the acrosome reaction are controversial. It has been shown that under certain conditions, PRG can stimulate the acrosome-associated processes, although there are significant differences between the data presented in the articles of different researchers [33,34]. Thus, in intact PRG-untreated bovine spermatozoa, capacitation activators (heparin, dbcAMP) increase the number of capacitated cells only during process of capacitation, and microfilaments are involved in this process. At the same time, an increase in the number of acrosome-reacted cells under the action of PRL occurred only when spermatozoa underwent the acrosome reaction with intact microtubules. In intact (PRG-untreated) spermatozoa of bulls, PRL did not promote processes of acrosome reaction during capacitation, while in sperm cells exposed with PRG, PRL ac- tivated the acrosome reaction in capacitated spermatozoa. In intact bovine spermatozoa, the effect of dbcAMP at the stage of the acrosome reaction had no effect on proportions of spermatozoa with different functional status, whereas in the presence of PRG, dbcAMP increased the percent of cells on capacitation stage. The action of dbcAMP during capacita- tion in untreated sperm cells was associated with microfilaments; the effect of PRL during the acrosome reaction was mediated by microtubules. At the same time, the effect of PRL at the stage of capacitation and the effect of dbcAMP during the acrosome reaction, in the presence of PRG, depended only on the intact microtubules. Thus, in bovine spermatozoa treated with PRG, the effects of dbcAMP and PRL did not depend on functional status of male gametes (capacitation or acrosome reaction). In this case (treatment with PRG) dbcAMP always (both at the capacitation stage and at the stage of acrosome reaction) increased the percent of capacitated spermatozoa, while PRL always (at both stages) stimulated the growth in the proportion of acrosome-reacted spermatozoa. After thawing bull sperm cells, their fertility is significantly reduced. The lower fertility of cryopreserved semen is partly due to premature capacitation-like modifications in considerable proportion of the sperm that affects the sample quality [35]. It has also been shown that cryopreservation procedures destroys microfilaments in mammalian cells and does not affect microtubules [36]. This finding raises the question of what the possible mechanism is for showing additional capacitated sperm cells after thawing. This increase likely occurs through the “intracellular mechanisms”, potentially due to the transformation of spermatozoa from capacitation stage to acrosome-reacted. In the presence of PRG in fresh bovine spermatozoa, all compounds, which activate processes of capacitation (heparin and Animals 2021, 11, 1788 9 of 11

Animals 2021, 11, x 10 of 12 dbcAMP) and acrosome reaction (prolactin), act through the microtubules, which remain intact even after thawing (Figure7).

Figure 7. Figure 7. TheThe effect effect of of progesterone progesterone on on functiona functionall status of native bovine bovine spermatozoa. spermatozoa. In In the the presence presence of of progesterone progesterone in in nativenative bovine spermatozoa,spermatozoa, dbcAMPdbcAMP and and heparin heparin induce induce the the process process of capacitationof capacitation in spermatozoain spermatozoa at the at acrosomethe acrosome reaction re- actionstage, whilestage, prolactin while prolactin stimulate stimulate the acrosome the acrosome reaction in reaction capacitated in cap cells;acitated both reactionscells; both go reactions through thego intactthrough microtubules. the intact microtubules. 5. Conclusions 5. ConclusionsIt can be assumed that, in thawed cell samples, an increase in the number of capacitated spermatozoaIt can be assumed occurs due that, to in the thawed transition cell ofsamples, acrosome-reacted an increase cellsin the to number capacitated of capaci- ones. tatedAccording spermatozoa to our results occurs obtained due to the in fresh transition sperm of cells, acrosome the induction-reacted of cells acrosome to capacitated reaction onein capacitateds. According sperm to our cells results in thawed obtained samples in fresh may sperm be mediated cells, the byinduction the action of ofacrosome PRG or reactionanother in compound capacitated acting sperm in acells similar in thawed manner. samples Furthermore, may be mediated this cell transformation by the action of is PRGassociated or another with thecompound functioning acting of cytoskeleton.in a similar manner. Furthermore, this cell transfor- mation is associated with the functioning of cytoskeleton. Author Contributions: Conceptualization, V.D. and T.K.; methodology, V.D., I.C., B.I.; software, I.C.; validation, V.D., I.C., B.I.; formal analysis, N.V., L.V.; investigation, V.D., I.C.; resources, B.I.; data Author Contributions: Conceptualization, V.D. and T.K.; methodology, V.D., I.C., B.I.; software, I.C.;curation, validation, T.K., N.V.; V.D., writing—original I.C., B.I.; formal draftanalysis, preparation, N.V., L.V.; V.D., investigation, I.C.; writing—review V.D., I.C.; and resource editing,s, T.K.,B.I.; dataN.V., curation, L.V., I.C.; T.K., visualization, N.V.; writing L.V.;— supervision,original draft T.K., preparation, N.V.; project V.D., administration, I.C.; writing— T.K.,review N.V.; and funding edit- ing,acquisition, T.K., N.V., T.K. L.V., All authorsI.C.; visualization, have read and L.V.; agreed supervision, to the published T.K., N.V.; version project of administration, the manuscript. T.K., N.V.;Funding: fundingThis acquisition, work was supported T.K. All authors by the Ministry have read of Scienceand agreed and Higherto the published Education version of the Russian of the manuscript.Federation(grant number No. 0445-2021-0005, 121052600350-9). Funding:Institutional This Review work was Board supported Statement: by theThe Ministry study wasof Science conducted and inHigher accordance Education with of the the guidelines Russian Federationof the Declaration (grant number of Helsinki No. and0445 in-2021 accordance-0005, 121052600350 with the Russian-9). “Guidelines for accommodation Institutionalof Animals 2021, Review 11, 189Board 8 of Statement: 9 and care The of laboratorystudy was animals.conducted Rules in accordance for keeping with and the care guidelines of farm ofanimals” the Declaration (State Standard-GOST-34088-2017) of Helsinki and in accordance dated with 12 Decemberthe Russian 2017. “Guidelines for accommodation ofInformed Animals Consent 2021, 11, Statement: 189 8 of 9 andNot care applicable. of laboratory animals. Rules for keeping and care of farm animals” (State Standard-GOST-34088-2017) dated 12 December 2017. Data Availability Statement: Not applicable. Informed Consent Statement: Not applicable. Acknowledgments: The authors wish to sincerely thank the collective of breeding farm OJSC DataNevskoe Availability for help inStatement: providing Not biological applicable. samples (semen of bulls) for present research. Acknowledgments:Conflicts of Interest: TheThe authors authors wishdeclare to no sincerely conflict ofthank interest. the collective of breeding farm OJSC Nevskoe for help in providing biological samples (semen of bulls) for present research. References Conflicts of Interest: The authors declare no conflict of interest. 1. Pons-Rejraji, H.; Bailey, J.L.; Leclerc, P. Modulation of bovine sperm signalling pathways: Correlation between intracellular Referencesparameters and sperm capacitation and acrosome exocytosis. Reprod. Fertil. Dev. 2009, 21, 511–524. [CrossRef] 2. 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